ActRII receptor polypeptides, methods and compositions

ABSTRACT

In certain aspects, the present invention provides compositions and methods for modulating (promoting or inhibiting) growth of a tissue, such as bone, cartilage, muscle, fat, and/or neuron. The present invention also provides methods of screening compounds that modulate activity of an ActRII protein and/or an ActRII ligand. The compositions and methods provided herein are useful in treating diseases associated with abnormal activity of an ActRII protein and/or an ActRII ligand.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/590,765, filed Jul. 23, 2004. All the teachings of theabove-referenced application is incorporated herein by reference.

BACKGROUND OF THE INVENTION

The transforming growth factor-beta (TGF-beta) superfamily contains avariety of growth factors that share common sequence elements andstructural motifs. These proteins are known to exert biological effectson a large variety of cell types in both vertebrates and invertebrates.Members of the superfamily perform important functions during embryonicdevelopment in pattern formation and tissue specification and caninfluence a variety of differentiation processes, includingadipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis,neurogenesis, and epithelial cell differentiation. The family is dividedinto two general branches: the BMP/GDF and the TGF-beta/Activin/BMP10branches, whose members have diverse, often complementary effects. Bymanipulating the activity of a member of the TGF-beta family, it isoften possible to cause significant physiological changes in anorganism. For example, the Piedmontese and Belgian Blue cattle breedscarry a loss-of-function mutation in the GDF8 (also called myostatin)gene that causes a marked increase in muscle mass. Grobet et al., NatGenet. 1997, 17(1):71-4. Furthermore, in humans, inactive alleles ofGDF8 are associated with increased muscle mass and, reportedly,exceptional strength. Schuelke et al., N Engl J Med 2004, 350:2682-8.

Changes in muscle, bone, cartilage and other tissues may be achieved byagonizing or antagonizing signaling that is mediated by an appropriateTGF-beta family member. Thus, there is a need for agents that functionas potent regulators of TGF-beta signaling.

SUMMARY OF THE INVENTION

In certain aspects, the present disclosure provides ActRII polypeptides.Such ActRII polypeptides may be used for the treatment of a variety ofdisorders or conditions, in particular, muscle and neuromusculardisorders (e.g., muscular dystrophy, amyotrophic lateral sclerosis(ALS), and muscle atrophy), undesired bone/cartilage growth, adiposetissue disorders (e.g., obesity), metabolic disorders (e.g., type 2diabetes), and neurodegenerative disorders. In specific embodiments,ActRII polypeptides (e.g., soluble ActRII polypeptides) can antagonizean ActRII receptor (e.g., ActRIIA or ActRIIB) generally, in any processassociated with ActRII activity. Optionally, ActRII polypeptides of theinvention may be designed to preferentially antagonize one or moreligands of ActRII receptors, such as GDF8 (also called myostatin),GDF11, activin, Nodal, and BMP7 (also called OP-1), and may therefore beuseful in the treatment of additional disorders. Examples of ActRIIpolypeptides include the naturally occurring ActRII polypeptides as wellas functional variants thereof.

In certain aspects, the disclosure provides pharmaceutical preparationscomprising a soluble ActRII (e.g., ActRIIA or ActRIIB) polypeptide thatbinds to an ActRII ligand such as GDF8, GDF11, activin, BMP7 or nodal,and a pharmaceutically acceptable carrier. Optionally, the solubleActRII polypeptide binds to an ActRII ligand with a Kd less than 10micromolar or less than 1 micromolar, 100, 10 or 1 nanomolar.Optionally, the soluble ActRII polypeptide inhibits ActRII signaling,such as intracellular signal transduction events triggered by an ActRIIligand. A soluble ActRII polypeptide for use in such a preparation maybe any of those disclosed herein, such as a polypeptide having an aminoacid sequence selected from SEQ ID NOs: 1-2 and 9-12, or having an aminoacid sequence that is at least 80%, 85%, 90%, 95%, 97% or 99% identicalto an amino acid sequence selected from SEQ ID NOs: 1-2 and 9-12. Asoluble ActRII polypeptide may include a functional fragment of anatural ActRII polypeptide, such as one comprising at least 10, 20 or 30amino acids of a sequence selected from SEQ ID NOs: 1-4 and 9-12 or asequence of SEQ ID NOs: 1 or 2, lacking the C-terminal 10 to 15 aminoacids (the “tail”). A soluble ActRII polypeptide may include one or morealterations in the amino acid sequence (e.g., in the ligand-bindingdomain) relative to a naturally occurring ActRII polypeptide. Thealteration in the amino acid sequence may, for example, alterglycosylation of the polypeptide when produced in a mammalian, insect orother eukaryotic cell or alter proteolytic cleavage of the polypeptiderelative to the naturally occurring ActRII polypeptide. A soluble ActRIIpolypeptide may be a fusion protein that has, as one domain, an ActRIIpolypeptide (e.g., a ligand-binding domain of an ActRII) and one or moreadditional domains that provide a desirable property, such as improvedpharmacokinetics, easier purification, targeting to particular tissues,etc.

For example, a domain of a fusion protein may enhance one or more of invivo stability, in vivo half life, uptake/administration, tissuelocalization or distribution, formation of protein complexes,multimerization of the fusion protein, and/or purification. A solubleActRII fusion protein may include an immunoglobulin Fc domain (wild-typeor mutant) or a serum albumin. In a preferred embodiment, an ActRII-Fcfusion comprises a relatively unstructured linker positioned between theFc domain and the extracellular ActRII domain. This unstructured linkermay correspond to the roughly 15 amino acid unstructured region at theC-terminal end of the extracellular domain of ActRIIA or ActRIIB (the“tail”), or it may be an artificial sequence of between 5 and 15, 20,30, 50 or more amino acids that are relatively free of secondarystructure. A linker may be rich in glycine and proline residues and may,for example, contain repeating sequences of threonine/serine andglycines (e.g., TG₄ or SG₄ repeats). A fusion protein may include apurification subsequence, such as an epitope tag, a FLAG tag, apolyhistidine sequence, and a GST fusion. Optionally, a soluble ActRIIpolypeptide includes one or more modified amino acid residues selectedfrom: a glycosylated amino acid, a PEGylated amino acid, a farnesylatedamino acid, an acetylated amino acid, a biotinylated amino acid, anamino acid conjugated to a lipid moiety, and an amino acid conjugated toan organic derivatizing agent. A pharmaceutical preparation may alsoinclude one or more additional compounds such as a compound that is usedto treat an ActRII-associated disorder. Preferably, a pharmaceuticalpreparation is substantially pyrogen free. In general, it is preferablethat an ActRII protein be expressed in a mammalian cell line thatmediates suitably natural glycosylation of the ActRII protein so as todiminish the likelihood of an unfavorable immune response in a patient.Human and CHO cell lines have been used successfully, and it is expectedthat other common mammalian expression vectors will be useful.

In certain aspects, the disclosure provides packaged pharmaceuticalscomprising a pharmaceutical preparation described herein and labeled foruse in promoting growth of a tissue or diminishing or preventing a lossof a tissue in a human. Exemplary tissues include bone, cartilage,muscle, fat, and neuron.

In certain aspects, the disclosure provides soluble ActRII polypeptidescomprising an altered ligand-binding (e.g., GDF8-binding) domain of anActRII. Such altered ligand-binding domains of an ActRII receptorcomprise one or more mutations at amino acid residues such as E37, E39,R40, K55, R56, Y60, A64, K74, W78, L79, D80, F82 and F101 of humanActRIIB. Such altered ligand-binding domains of an ActRII receptorcomprise one or more mutations at amino acid residues such as E38, E40,R41, K56, R57, Y61, K65, K75, W79, L80, D81, 183 and F102 of humanActRIIA. Optionally, the altered ligand-binding domain can haveincreased selectivity for a ligand such as GDF8/GDF11 relative to awild-type ligand-binding domain of an ActRII receptor. To illustrate,these mutations are demonstrated herein to increase the selectivity ofthe altered ligand-binding domain for GDF11 (and therefore, presumably,GDF8) over activin (presented with respect to ActRIIB): K74Y, K74F, K74Iand D80I. The following mutations have the reverse effect, increasingthe ratio of activin binding over GDF11: D54A, K55A, L79A and F82A. Theoverall (GDF11 and activin) binding activity can be increased byinclusion of the “tail” region or, presumably, a unstructured linkerregion, and also by use of a mutation such as A64R (which occursnaturally) or K74A. Other mutations that caused an overall decrease inligand binding affinity, include: R40A, E37A, R56A, W78A, D80K, D80R,D80A, D80G, D80F, D80M and D80N. Mutations may be combined to achievedesired effects. For example, many of the mutations that affect theratio of GDF11:Activin binding have an overall negative effect on ligandbinding, and therefore, these may be combined with mutations thatgenerally increase ligand binding to produce an improved binding proteinwith ligand selectivity.

Optionally, the altered ligand-binding domain has a ratio of K_(d) foractivin binding to K_(d) for GDF8 binding that is at least 2, 5, 10, oreven 100 fold greater relative to the ratio for the wild-typeligand-binding domain. Optionally, the altered ligand-binding domain hasa ratio of IC₅₀ for inhibiting activin to IC₅₀ for inhibiting GDF8/GDF11that is at least 2, 5, 10, or even 100 fold greater relative to thewild-type ligand-binding domain. Optionally, the altered ligand-bindingdomain inhibits GDF8/GDF11 with an IC₅₀ at least 2, 5, 10, or even 100times less than the IC₅₀ for inhibiting activin. These soluble ActRIIpolypeptides can be fusion proteins that include an immunoglobulin Fcdomain (either wild-type or mutant). In certain cases, the subjectsoluble ActRII polypeptides are antagonists (inhibitors) of GDF8/GDF11.

In certain aspects, the disclosure provides nucleic acids encoding asoluble ActRII polypeptide, which do not encode a complete ActRIIpolypeptide. An isolated polynucleotide may comprise a coding sequencefor a soluble ActRII polypeptide, such as described above. For example,an isolated nucleic acid may include a sequence coding for anextracellular domain (e.g., ligand-binding domain) of an ActRII and asequence that would code for part or all of the transmembrane domainand/or the cytoplasmic domain of an ActRII, but for a stop codonpositioned within the transmembrane domain or the cytoplasmic domain, orpositioned between the extracellular domain and the transmembrane domainor cytoplasmic domain. For example, an isolated polynucleotide maycomprise a full-length ActRII polynucleotide sequence such as SEQ ID NO:7 or 8, or a partially truncated version, said isolated polynucleotidefurther comprising a transcription termination codon at least sixhundred nucleotides before the 3′-terminus or otherwise positioned suchthat translation of the polynucleotide gives rise to an extracellulardomain optionally fused to a truncated portion of a full-length ActRII.Nucleic acids disclosed herein may be operably linked to a promoter forexpression, and the disclosure provides cells transformed with suchrecombinant polynucleotides. Preferably the cell is a mammalian cellsuch as a CHO cell.

In certain aspects, the disclosure provides methods for making a solubleActRII polypeptide. Such a method may include expressing any of thenucleic acids (e.g., SEQ ID NO: 5 or 6) disclosed herein in a suitablecell, such as a Chinese hamster ovary (CHO) cell. Such a method maycomprise: a) culturing a cell under conditions suitable for expressionof the soluble ActRII polypeptide, wherein said cell is transformed witha soluble ActRII expression construct; and b) recovering the solubleActRII polypeptide so expressed. Soluble ActRII polypeptides may berecovered as crude, partially purified or highly purified fractionsusing any of the well known techniques for obtaining protein from cellcultures.

In certain aspects, a soluble ActRII polypeptide disclosed herein may beused in a method for treating a subject having a disorder associatedwith muscle loss or insufficient muscle growth. Such disorders includemuscle atrophy, muscular dystrophy, amyotrophic lateral sclerosis (ALS),and a muscle wasting disorder (e.g., cachexia, anorexia, DMD syndrome,BMD syndrome, AIDS wasting syndrome, muscular dystrophies, neuromusculardiseases, motor neuron diseases, diseases of the neuromuscular junction,and inflammatory myopathies). A method may comprise administering to asubject in need thereof an effective amount of a soluble ActRIIpolypeptide.

In certain aspects, a soluble ActRII polypeptide disclosed herein may beused in a method for treating a subject having a disorder associatedwith neurodegeneration. Such disorders include Alzheimer's Disease (AD),Parkinson's Disease (PD), Amyotrophic Lateral Sclerosis (ALS),Huntington's disease (HD). A method may comprise administering to asubject in need thereof an effective amount of a soluble ActRIIpolypeptide.

In certain aspects, a soluble ActRII polypeptide disclosed herein may beused in a method for treating a subject having a disorder associatedwith abnormal cell growth and differentiation. Such disorders includeinflammation, allergy, autoimmune diseases, infectious diseases, andtumors. A method may comprise administering to a subject in need thereofan effective amount of a soluble ActRII polypeptide. A selective activinbinding ActRII protein may be particularly useful for treating anactivin-dependent cancer, such as ovarian cancer.

In certain aspects, a soluble ActRII polypeptide disclosed herein may beused in a method for decreasing the body fat content or reducing therate of increase in body fat content, and for treating a disorderassociated with undesirable body weight gain, such as obesity,non-insulin dependent diabetes mellitus (NIDDM), cardiovascular disease,cancer, hypertension, osteoarthritis, stroke, respiratory problems, andgall bladder disease. These methods may comprise administering to asubject in need thereof an effective amount of a soluble ActRIIpolypeptide.

In certain specific aspects, a soluble ActRII polypeptide disclosedherein may be used in a method for treating a disorder associated withabnormal activity of GDF8. Such disorders include metabolic disorderssuch as type 2 diabetes, impaired glucose tolerance, metabolic syndrome(e.g., syndrome X), and insulin resistance induced by trauma (e.g.,burns or nitrogen imbalance); adipose tissue disorders (e.g., obesity);muscular dystrophy (including Duchenne's muscular dystrophy);amyotrophic lateral sclerosis (ALS); muscle atrophy; organ atrophy;frailty; carpal tunnel syndrome; congestive obstructive pulmonarydisease; sarcopenia, cachexia and other muscle wasting syndromes;osteoporosis; glucocorticoid-induced osteoporosis; osteopenia;osteoarthritis; osteoporosis-related fractures; low bone mass due tochronic glucocorticoid therapy, premature gonadal failure, androgensuppression, vitamin D deficiency, secondary hyperparathyroidism,nutritional deficiencies, and anorexia nervosa. The method may compriseadministering to a subject in need thereof an effective amount of asoluble ActRII polypeptide.

In certain aspects, the disclosure provides a method for identifying anagent that stimulates growth of a tissue such as bone, cartilage,muscle, fat, and neuron. The method comprises: a) identifying a testagent that binds to a ligand-binding domain of an ActRII polypeptidecompetitively with a soluble ActRII polypeptide; and b) evaluating theeffect of the agent on growth of the tissue.

In certain aspects, the disclosure provides methods for antagonizingactivity of an ActRII polypeptide or an ActRII ligand (e.g., GDF8,GDF11, activin, BMP7, and Nodal) in a cell. The methods comprisecontacting the cell with a soluble ActRII polypeptide. Optionally, theactivity of the ActRII polypeptide or the ActRII ligand is monitored bya signaling transduction mediated by the ActRII/ActRII ligand complex,for example, by monitoring cell proliferation. The cells of the methodsinclude an osteoblast, a chondrocyte, a myocyte, an adipocyte, a musclecell, and a neuronal cell.

In certain aspects, the disclosure provides uses of a soluble ActRIIpolypeptide for making a medicament for the treatment of a disorder orcondition as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a human ActRIIA soluble (extracellular) polypeptidesequence (SEQ ID NO: 1). The C-terminal “tail” is underlined.

FIG. 2 shows a human ActRIIB soluble (extracellular) polypeptidesequence (SEQ ID NO: 2). The C-terminal “tail” is underlined.

FIG. 3 shows human ActRIIA precursor protein sequence (SEQ ID NO: 3).The signal peptide is underlined; the extracellular domain is in bold(also referred to as SEQ ID NO: 1); and the potential N-linkedglycosylation sites are boxed.

FIG. 4 shows human ActRIIB precursor protein sequence (SEQ ID NO: 4).The signal peptide is underlined; the extracellular domain is in bold(also referred to as SEQ ID NO: 2); and the potential N-linkedglycosylation sites are boxed.

FIG. 5 shows a nucleic acid sequence encoding a human ActRIIA soluble(extracellular) polypeptide, designed as SEQ ID NO: 5.

FIG. 6 shows a nucleic acid sequence encoding a human ActRIIB soluble(extracellular) polypeptide, designed as SEQ ID NO: 6.

FIG. 7 shows a nucleic acid sequence encoding human ActRIIA precursorprotein, designed as SEQ ID NO: 7.

FIG. 8 shows a nucleic acid sequence encoding human ActRIIB precursorprotein, designed as SEQ ID NO: 8.

FIG. 9 shows expression of the extracellular (soluble) domains ofActRIIA or ActRIIB. Constructs expressing human extracellular domains ofActRIIA or ActRIIB were made with all three signal sequences.

FIG. 10 shows three soluble ActRIIB polypeptides with various signalsequences, SEQ ID NOs: 9-11.

FIG. 11 shows one soluble ActRIIA polypeptide with its native signalsequence, SEQ ID NO: 12.

FIG. 12 shows design of the Fc fusions of ActRIIA or ActRIIBpolypeptides. The flexible linker sequence and the Fc sequence (SEQ IDNO: 13) are shown. Mutations can be made at one more amino acid residuesof the Fc sequence. Examples of such residues for mutations areunderlined, and referred to as Asp-265, lysine-322, and Asn-434.

FIG. 13 shows the ligand-binding pocket of an ActRIIB polypeptide.Examples of amino acid residues in the ligand-binding pocket are shownas E39, K55, Y60, K74, W78, D80, and F101. ActRIIB polypeptides of theinvention may comprise mutations at one or more of these amino acidresidues.

FIG. 14 shows an alignment of the extracellular domains of ActRIIA andActRIIB, with the positions of mutations that, in ActRIIB, aredemonstrated herein to affect ligand binding. The alignment shows thatthe position of these mutations is conserved in ActRIIA.

FIG. 15 shows a schematic for the A-204 Reporter Gene Assay. The figureshows the Reporter vector: pGL3(CAGA)12 (described in Dennler et al,1998, EMBO 17: 3091-3100.) The CAGA12 motif is present in TGF-Betaresponsive genes (PAI-1 gene), so this vector is of general use forfactors signaling through Smad2 and 3.

FIG. 16 shows the effects of various mutations in ActRIIB-Fc on a GDF-11A-204 Reporter Gene Assay. The background A64 construct showed the leasteffect on GDF-11 activity. The A64K mutation (also a naturally occurringform) caused a substantial increase in GDF-11 inhibition, and acombination of the A64K mutation with the addition of the 15 C-terminalamino acids of the extracellular domain (the 15 amino acid “tail”)produced an even more potent inhibitor of GDF-11 activity.

FIG. 17 shows the effects of various mutations in ActRIIB-Fc on anActivin A, A-204 Reporter Gene Assay. The background A64 constructshowed the least effect on Activin A activity. The K74A mutation causeda substantial increase in Activin A inhibition. A control sample lackingActivin A showed no activity.

DETAILED DESCRIPTION OF THE INVENTION

1. Overview

In certain aspects, the present invention relates to ActRIIpolypeptides. As used herein, the term “ActRII” refers to a family ofactivin receptor type II (ActRII) proteins and ActRII-related proteins,derived from any species. Reference to ActRII herein is understood to bea reference to any one of the currently identified forms, includingActRIIA (also known as ActRII) and ActRIIB. Members of the ActRII familyare generally all transmembrane proteins, composed of a ligand-bindingextracellular domain with cysteine-rich region, a transmembrane domain,and a cytoplasmic domain with predicted serine/threonine kinasespecificity. Amino acid sequences of human ActRIIA precursor protein andActRIIB precursor protein are illustrated in FIG. 3 (SEQ ID NO: 3) andFIG. 4 (SEQ ID NO: 4), respectively.

The term “ActRII polypeptide” is used to refer to polypeptidescomprising any naturally occurring polypeptide of an ActRII familymember as well as any variants thereof (including mutants, fragments,fusions, and peptidomimetic forms) that retain a useful activity. Forexample, ActRII polypeptides include polypeptides derived from thesequence of any known ActRII having a sequence at least about 80%identical to the sequence of an ActRII polypeptide, and preferably atleast 85%, 90%, 95%, 97%, 99% or greater identity.

In a specific embodiment, the invention relates to soluble ActRIIpolypeptides. As described herein, the term “soluble ActRII polypeptide”generally refers to polypeptides comprising an extracellular domain ofan ActRII protein. The term “soluble ActRII polypeptide,” as usedherein, includes any naturally occurring extracellular domain of anActRII protein as well as any variants thereof (including mutants,fragments and peptidomimetic forms) that retain a useful activity. Forexample, the extracellular domain of an ActRII protein binds to a ligandand is generally soluble. Examples of soluble ActRII polypeptidesinclude ActRIIA and ActRIIB soluble polypeptides illustrated in FIG. 1(SEQ ID NO: 1) and FIG. 2 (SEQ ID NO: 2), respectively. Other examplesof soluble ActRII polypeptides comprise a signal sequence in addition tothe extracellular domain of an ActRII protein, for example, thesequences illustrated in FIG. 10 (SEQ ID NOs: 9-11) and FIG. 11 (SEQ IDNO: 12). The signal sequence can be a native signal sequence of anActRII, or a signal sequence from another protein, such as a tissueplasminogen activator (TPA) signal sequence or a honey bee melatin (HBM)signal sequence.

TGF-β signals are mediated by heteromeric complexes of type I and typeII serine/threonine kinase receptors, which phosphorylate and activatedownstream Smad proteins upon ligand stimulation (Massagué, 2000, Nat.Rev. Mol. Cell Biol. 1:169-178). These type I and type II receptors areall transmembrane proteins, composed of a ligand-binding extracellulardomain with cysteine-rich region, a transmembrane domain, and acytoplasmic domain with predicted serine/threonine specificity. Type Ireceptors are essential for signaling; and type II receptors arerequired for binding ligands and for expression of type I receptors.Type I and II activin receptors form a stable complex after ligandbinding, resulting in phosphorylation of type I receptors by type IIreceptors.

Two related type II receptors, ActRIIA and ActRIIB, have been identifiedas the type II receptors for activins (Mathews and Vale, 1991, Cell65:973-982; Attisano et al., 1992, Cell 68: 97-108). Besides activins,ActRIIA and ActRIIB can biochemically interact with several other TGF-βfamily proteins, including BMP7, Nodal, GDF8, and GDF11 (Yamashita etal., 1995, J. Cell Biol. 130:217-226; Lee and McPherron, 2001, Proc.Natl. Acad. Sci. 98:9306-9311; Yeo and Whitman, 2001, Mol. Cell 7:949-957; Oh et al., 2002, Genes Dev. 16:2749-54).

In certain embodiments, the present invention relates to antagonizing aligand of ActRII receptors (also referred to as an ActRII ligand) with asubject ActRII polypeptide (e.g., a soluble ActRII polypeptide). Thus,compositions and methods of the present invention are useful fortreating disorders associated with abnormal activity of one or moreligands of ActRII receptors. Exemplary ligands of ActRII receptorsinclude some TGF-β family members, such as activin, Nodal, GDF8, GDF11,and BMP7. These ligands of ActRII receptors are described in more detailbelow.

Activins are dimeric polypeptide growth factors and belong to theTGF-beta superfamily. There are three activins (A, B, and AB) that arehomo/heterodimers of two closely related β subunits (β_(A)β_(A),β_(B)β_(B), and β_(A)β_(B)). In the TGF-beta superfamily, activins areunique and multifunctional factors that can stimulate hormone productionin ovarian and placental cells, support neuronal cell survival,influence cell-cycle progress positively or negatively depending on celltype, and induce mesodermal differentiation at least in amphibianembryos (DePaolo et al., 1991, Proc SocEp Biol Med. 198:500-512; Dysonet al., 1997, Curr Biol. 7:81-84; Woodruff, 1998, Biochem Pharmacol.55:953-963). Moreover, erythroid differentiation factor (EDF) isolatedfrom the stimulated human monocytic leukemic cells was found to beidentical to activin A (Murata et al., 1988, PNAS, 85:2434). It wassuggested that activin A acts as a natural regulator of erythropoiesisin the bone marrow. In several tissues, activin signaling is antagonizedby its related heterodimer, inhibin. For example, during the release offollicle-stimulating hormone (FSH) from the pituitary, activin promotesFSH secretion and synthesis, while inhibin prevents FSH secretion andsynthesis. Other proteins that may regulate activin bioactivity and/orbind to activin include follistatin (FS), follistatin-related protein(FSRP), α₂-macroglobulin, Cerberus, and endoglin, which are describedbelow.

Nodal proteins have functions in mesoderm and endoderm induction andformation, as well as subsequent organization of axial structures suchas heart and stomach in early embryogenesis. It has been demonstratedthat dorsal tissue in a developing vertebrate embryo contributespredominantly to the axial structures of the notochord and pre-chordalplate while it recruits surrounding cells to form non-axial embryonicstructures. Nodal appears to signal through both type I and type IIreceptors and intracellular effectors known as Smad proteins. Recentstudies support the idea that ActRIIA and ActRIIB serve as type IIreceptors for Nodal (Sakuma et al., Genes Cells. 2002, 7:401-12). It issuggested that Nodal ligands interact with their co-factors (e.g.,cripto) to activate activin type I and type II receptors, whichphosphorylate Smad2. Nodal proteins are implicated in many eventscritical to the early vertebrate embryo, including mesoderm formation,anterior patterning, and left-right axis specification. Experimentalevidence has demonstrated that Nodal signaling activates pAR3-Lux, aluciferase reporter previously shown to respond specifically to activinand TGF-beta. However, Nodal is unable to induce pTlx2-Lux, a reporterspecifically responsive to bone morphogenetic proteins. Recent resultsprovide direct biochemical evidence that Nodal signaling is mediated byboth activin-TGF-beta pathway Smads, Smad2 and Smad3. Further evidencehas shown that the extracellular cripto protein is required for Nodalsignaling, making it distinct from activin or TGF-beta signaling.

Growth and Differentiation Factor-8 (GDF8) is also known as myostatin.GDF8 is a negative regulator of skeletal muscle mass. GDF8 is highlyexpressed in the developing and adult skeletal muscle. The GDF8 nullmutation in transgenic mice is characterized by a marked hypertrophy andhyperplasia of the skeletal muscle (McPherron et al., Nature, 1997,387:83-90). Similar increases in skeletal muscle mass are evident innaturally occurring mutations of GDF8 in cattle (Ashmore et al., 1974,Growth, 38:501-507; Swatland and Kieffer, J. Anim. Sci., 1994,38:752-757; McPherron and Lee, Proc. Natl. Acad. Sci. USA, 1997,94:12457-12461; and Kambadur et al., Genome Res., 1997, 7:910-915) and,strikingly, in humans (Schuelke et al., N Engl J Med 2004; 350:2682-8).Studies have also shown that muscle wasting associated withHIV-infection in humans is accompanied by increases in GDF8 proteinexpression (Gonzalez-Cadavid et al., PNAS, 1998, 95:14938-43). Inaddition, GDF8 can modulate the production of muscle-specific enzymes(e.g., creatine kinase) and modulate myoblast cell proliferation (WO00/43781). The GDF8 propeptide can noncovalently bind to the mature GDF8domain dimer, inactivating its biological activity (Miyazono et al.(1988) J. Biol. Chem., 263: 6407-6415; Wakefield et al. (1988) J. Biol.Chem., 263; 7646-7654; and Brown et al. (1990) Growth Factors, 3:35-43). Other proteins which bind to GDF8 or structurally relatedproteins and inhibit their biological activity include follistatin, andpotentially, follistatin-related proteins (Gamer et al. (11999) Dev.Biol., 208: 222-232).

Growth and Differentiation Factor-11 (GDF11), also known as BMP11, is asecreted protein (McPherron et al., 1999, Nat. Genet. 22: 260-264).GDF11 is expressed in the tail bud, limb bud, maxillary and mandibulararches, and dorsal root ganglia during mouse development (Nakashima etal., 1999, Mech. Dev. 80: 185-189). GDF11 plays a unique role inpatterning both mesodermal and neural tissues (Gamer et al., 1999, DevBiol., 208:222-32). GDF11 was shown to be a negative regulator ofchondrogenesis and myogenesis in developing chick limb (Gamer et al.,2001, Dev Biol. 229:407-20). The expression of GDF11 in muscle alsosuggests its role in regulating muscle growth in a similar way to GDF8.In addition, the expression of GDF11 in brain suggests that GDF11 mayalso possess activities that relate to the function of the nervoussystem. Interestingly, GDF11 was found to inhibit neurogenesis in theolfactory epithelium (Wu et al., 2003, Neuron. 37:197-207). Hence, GDF11may have in vitro and in vivo applications in the treatment of diseasessuch as muscle diseases and neurodegenerative diseases (e.g.,amyotrophic lateral sclerosis). Bone morphogenetic protein (BMP7), alsocalled osteogenic protein-1 (OP-1), is well known to induce cartilageand bone formation. In addition, BMP7 regulates a wide array ofphysiological processes. For example, BMP7 may be the osteoinductivefactor responsible for the phenomenon of epithelial osteogenesis. It isalso found that BMP7 plays a role in calcium regulation and bonehomeostasis. Like activin, BMP7 binds to type II receptors, ActRIIA andIIB. However, BMP7 and activin recruit distinct type I receptors intoheteromeric receptor complexes. The major BMP7 type I receptor observedwas ALK2, while activin bound exclusively to ALK4 (ActRIIB). BMP7 andactivin elicited distinct biological responses and activated differentSmad pathways (Macias-Silva et al., 1998, J Biol. Chem. 273:25628-36).

In certain aspects, the present invention relates to the use of certainActRII polypeptides (e.g., soluble ActRII polypeptides) to antagonizeActRII receptors generally, in any process associated with ActRIIactivity. Optionally, ActRII polypeptides of the invention mayantagonize one or more ligands of ActRII receptors, such as activin,Nodal, GDF8, GDF11, and BMP7, and may therefore be useful in thetreatment of additional disorders.

Therefore, the present invention contemplates using ActRII polypeptidesin treating or preventing diseases or conditions that are associatedwith abnormal activity of an ActRII or an ActRII ligand. ActRII orActRII ligands are involved in the regulation of many criticalbiological processes. Due to their key functions in these processes,they may be desirable targets for therapeutic intervention. For example,ActRII polypeptides (e.g., e.g., soluble ActRII polypeptides) may beused to treat human or animal disorders or conditions. Example of suchdisorders or conditions include, but are not limited to, metabolicdisorders such as type 2 diabetes, impaired glucose tolerance, metabolicsyndrome (e.g., syndrome X), and insulin resistance induced by trauma(e.g., burns or nitrogen imbalance); adipose tissue disorders (e.g.,obesity); muscle and neuromuscular disorders such as muscular dystrophy(including Duchenne's muscular dystrophy); amyotrophic lateral sclerosis(ALS); muscle atrophy; organ atrophy; frailty; carpal tunnel syndrome;congestive obstructive pulmonary disease; and sarcopenia, cachexia andother muscle wasting syndromes. Other examples include osteoporosis,especially in the elderly and/or postmenopausal women;glucocorticoid-induced osteoporosis; osteopenia; osteoarthritis; andosteoporosis-related fractures. Yet further examples include low bonemass due to chronic glucocorticoid therapy, premature gonadal failure,androgen suppression, vitamin D deficiency, secondaryhyperparathyroidism, nutritional deficiencies, and anorexia nervosa.These disorders and conditions are discussed below under “ExemplaryTherapeutic Uses.”

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them. The scope or meaning of any useof a term will be apparent from the specific context in which the termis used.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Typically, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values.

Alternatively, and particularly in biological systems, the terms “about”and “approximately” may mean values that are within an order ofmagnitude, preferably within 5-fold and more preferably within 2-fold ofa given value. Numerical quantities given herein are approximate unlessstated otherwise, meaning that the term “about” or “approximately” canbe inferred when not expressly stated.

The methods of the invention may include steps of comparing sequences toeach other, including wild-type sequence to one or more mutants(sequence variants). Such comparisons typically comprise alignments ofpolymer sequences, e.g., using sequence alignment programs and/oralgorithms that are well known in the art (for example, BLAST, FASTA andMEGALIGN, to name a few). The skilled artisan can readily appreciatethat, in such alignments, where a mutation contains a residue insertionor deletion, the sequence alignment will introduce a “gap” (typicallyrepresented by a dash, or “A”) in the polymer sequence not containingthe inserted or deleted residue.

“Homologous,” in all its grammatical forms and spelling variations,refers to the relationship between two proteins that possess a “commonevolutionary origin,” including proteins from superfamilies in the samespecies of organism, as well as homologous proteins from differentspecies of organism. Such proteins (and their encoding nucleic acids)have sequence homology, as reflected by their sequence similarity,whether in terms of percent identity or by the presence of specificresidues or motifs and conserved positions.

The term “sequence similarity,” in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin.

However, in common usage and in the instant application, the term“homologous,” when modified with an adverb such as “highly,” may referto sequence similarity and may or may not relate to a commonevolutionary origin.

2. ActRII Polypeptides

In certain aspects, the invention relates to ActRII polypeptides (e.g.,soluble ActRII polypeptides). Preferably, the fragments, functionalvariants, and modified forms have similar or the same biologicalactivities of their corresponding wild-type ActRII polypeptides. Forexample, an ActRII polypeptide of the invention may bind to and inhibitfunction of an ActRII protein and/or an ActRII ligand protein (e.g.,activin, Nodal, GDF8, GDF11 or BMP7). Optionally, an ActRII polypeptidemodulates growth of tissues such as bone, cartilage, muscle, fat, and/orneuron. Examples of ActRII polypeptides include human ActRIIA precursorpolypeptide (SEQ ID NO: 3), human ActRIIB precursor polypeptide (SEQ IDNO: 4), soluble human ActRIIA polypeptides (e.g., SEQ ID NOs: 1 and 12),soluble human ActRIIB polypeptides (e.g., SEQ ID NOs: 2 and 9-11).

In certain embodiments, isolated fragments of the ActRII polypeptidescan be obtained by screening polypeptides recombinantly produced fromthe corresponding fragment of the nucleic acid encoding an ActRIIpolypeptide (e.g., one of SEQ ID NOs: 1-2 and 9-12). In addition,fragments can be chemically synthesized using techniques known in theart such as conventional Merrifield solid phase f-Moc or t-Bocchemistry. The fragments can be produced (recombinantly or by chemicalsynthesis) and tested to identify those peptidyl fragments that canfunction, for example, as antagonists (inhibitors) or agonists(activators) of an ActRII protein or an ActRII ligand.

In certain embodiments, a functional variant of the ActRII polypeptideshas an amino acid sequence that is at least 75% identical to an aminoacid sequence selected from SEQ ID NOs: 1-2 and 9-12. In certain cases,the functional variant has an amino acid sequence at least 80%, 85%,90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequenceselected from SEQ ID NOs: 1-2 and 9-12.

In certain embodiments, the present invention contemplates makingfunctional variants by modifying the structure of an ActRII polypeptidefor such purposes as enhancing therapeutic efficacy, or stability (e.g.,ex vivo shelf life and resistance to proteolytic degradation in vivo).Such modified ActRII polypeptides when designed to retain at least oneactivity of the naturally-occurring form of the ActRII polypeptides, areconsidered functional equivalents of the naturally-occurring ActRIIpolypeptides. Modified ActRII polypeptides can also be produced, forinstance, by amino acid substitution, deletion, or addition. Forinstance, it is reasonable to expect that an isolated replacement of aleucine with an isoleucine or valine, an aspartate with a glutamate, athreonine with a serine, or a similar replacement of an amino acid witha structurally related amino acid (e.g., conservative mutations) willnot have a major effect on the biological activity of the resultingmolecule. Conservative replacements are those that take place within afamily of amino acids that are related in their side chains. Whether achange in the amino acid sequence of an ActRII polypeptide results in afunctional homolog can be readily determined by assessing the ability ofthe variant ActRII polypeptide to produce a response in cells in afashion similar to the wild-type ActRII polypeptide.

In certain specific embodiments, the present invention contemplatesmaking mutations in the extracellular domain (also referred to asligand-binding domain) of an ActRII polypeptide such that the variant(or mutant) ActRII polypeptide has altered ligand-binding activities(e.g., binding affinity or binding specificity). In certain cases, suchvariant ActRII polypeptides have altered (elevated or reduced) bindingaffinity for a specific ligand. In other cases, the variant ActRIIpolypeptides have altered binding specificity for their ligands.

For example, the variant ActRII polypeptide preferentially binds to aspecific ligand (e.g., GDF8). For example, amino acid residues of theActRIIB protein, such as E39, K55, Y60, K74, W78, D80, and F101 (shownin FIG. 13), are in the ligand-binding pocket and mediate binding to itsligands such as activin and GDF8. Thus, the present invention providesan altered ligand-binding domain (e.g., GDF8-binding domain) of anActRII receptor, which comprises one or more mutations at those aminoacid residues. Optionally, the altered ligand-binding domain can haveincreased selectivity for a ligand such as GDF8 relative to a wild-typeligand-binding domain of an ActRII receptor. To illustrate, thesemutations increase the selectivity of the altered ligand-binding domainfor GDF8 over activin. Optionally, the altered ligand-binding domain hasa ratio of K_(d) for activin binding to K_(d) for GDF8 binding that isat least 2, 5, 10, or even 100 fold greater relative to the ratio forthe wild-type ligand-binding domain. Optionally, the alteredligand-binding domain has a ratio of IC₅₀ for inhibiting activin to IC₅₀for inhibiting GDF8 that is at least 2, 5, 10, or even 100 fold greaterrelative to the wild-type ligand-binding domain. Optionally, the alteredligand-binding domain inhibits GDF8 with an IC₅₀ at least 2, 5, 10, oreven 100 times less than the IC₅₀ for inhibiting activin.

As an specific example, the positively-charged amino acid residue Asp(D80) of the ligand-binding domain of ActRIIB can be mutated to adifferent amino acid residue such that the variant ActRII polypeptidepreferentially binds to GDF8, but not activin. Preferably, the D60residue is changed to an amino acid residue selected from the groupconsisting of: a uncharged amino acid residue, a negative amino acidresidue, and a hydrophobic amino acid residue. As will be recognized byone of skill in the art, most of the described mutations, variants ormodifications may be made at the nucleic acid level or, in some cases,by post translational modification or chemical synthesis. Suchtechniques are well known in the art.

In certain embodiments, the present invention contemplates specificmutations of the ActRII polypeptides so as to alter the glycosylation ofthe polypeptide. Exemplary glycosylation sites in ActRIIA and ActRIIBpolypeptides are illustrated in FIGS. 3 and 4 respectively. Suchmutations may be selected so as to introduce or eliminate one or moreglycosylation sites, such as O-linked or N-linked glycosylation sites.Asparagine-linked glycosylation recognition sites generally comprise atripeptide sequence, asparagine-X-threonine (where “X” is any aminoacid) which is specifically recognized by appropriate cellularglycosylation enzymes. The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the wild-type ActRII polypeptide (for O-linked glycosylationsites). A variety of amino acid substitutions or deletions at one orboth of the first or third amino acid positions of a glycosylationrecognition site (and/or amino acid deletion at the second position)results in non-glycosylation at the modified tripeptide sequence.Another means of increasing the number of carbohydrate moieties on anActRII polypeptide is by chemical or enzymatic coupling of glycosides tothe ActRII polypeptide. Depending on the coupling mode used, thesugar(s) may be attached to (a) arginine and histidine; (b) freecarboxyl groups; (c) free sulfhydryl groups such as those of cysteine;(d) free hydroxyl groups such as those of serine, threonine, orhydroxyproline; (e) aromatic residues such as those of phenylalanine,tyrosine, or tryptophan; or (f) the amide group of glutamine. Thesemethods are described in WO 87/05330 published Sep. 11, 1987, and inAplin and Wriston (1981) CRC Crit. Rev. Biochem., pp. 259-306,incorporated by reference herein. Removal of one or more carbohydratemoieties present on an ActRII polypeptide may be accomplished chemicallyand/or enzymatically. Chemical deglycosylation may involve, for example,exposure of the ActRII polypeptide to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving the aminoacid sequence intact. Chemical deglycosylation is further described byHakimuddin et al. (1987) Arch. Biochem. Biophys. 259:52 and by Edge etal. (1981) Anal. Biochem. 118:131. Enzymatic cleavage of carbohydratemoieties on ActRII polypeptides can be achieved by the use of a varietyof endo- and exo-glycosidases as described by Thotakura et al. (1987)Meth. Enzymol. 138:350. The sequence of an ActRII polypeptide may beadjusted, as appropriate, depending on the type of expression systemused, as mammalian, yeast, insect and plant cells may all introducediffering glycosylation patterns that can be affected by the amino acidsequence of the peptide. In general, ActRII proteins for use in humanswill be expressed in a mammalian cell line that provides properglycosylation, such as HEK293 or CHO cell lines, although othermammalian expression cell lines are expected to be useful as well.

This disclosure further contemplates a method of generating mutants,particularly sets of combinatorial mutants of an ActRII polypeptide, aswell as truncation mutants; pools of combinatorial mutants areespecially useful for identifying functional variant sequences. Thepurpose of screening such combinatorial libraries may be to generate,for example, ActRII polypeptide variants which can act as eitheragonists or antagonist, or alternatively, which possess novel activitiesall together. A variety of screening assays are provided below, and suchassays may be used to evaluate variants. For example, an ActRIIpolypeptide variant may be screened for ability to bind to an ActRIIpolypeptide, to prevent binding of an ActRII ligand to an ActRIIpolypeptide.

The activity of an ActRII polypeptide or its variants may also be testedin a cell-based or in vivo assay. For example, the effect of an ActRIIpolypeptide variant on the expression of genes involved in boneproduction in an osteoblast or precursor may be assessed. This may, asneeded, be performed in the presence of one or more recombinant ActRIIligand protein (e.g., BMP7), and cells may be transfected so as toproduce an ActRII polypeptide and/or variants thereof, and optionally,an ActRII ligand. Likewise, an ActRII polypeptide may be administered toa mouse or other animal, and one or more bone properties, such asdensity or volume may be assessed. The healing rate for bone fracturesmay also be evaluated. Similarly, the activity of an ActRII polypeptideor its variants may be tested in muscle cells, adipocytes, and neuroncells for any effect on growth of these cells, for example, by theassays as described below. Such assays are well known and routine in theart.

Combinatorially-derived variants can be generated which have a selectivepotency relative to a naturally occurring ActRII polypeptide. Suchvariant proteins, when expressed from recombinant DNA constructs, can beused in gene therapy protocols. Likewise, mutagenesis can give rise tovariants which have intracellular half-lives dramatically different thanthe corresponding a wild-type ActRII polypeptide. For example, thealtered protein can be rendered either more stable or less stable toproteolytic degradation or other cellular processes which result indestruction of, or otherwise inactivation of a native ActRIIpolypeptide. Such variants, and the genes which encode them, can beutilized to alter ActRII polypeptide levels by modulating the half-lifeof the ActRII polypeptides. For instance, a short half-life can giverise to more transient biological effects and, when part of an inducibleexpression system, can allow tighter control of recombinant ActRIIpolypeptide levels within the cell.

In a preferred embodiment, the combinatorial library is produced by wayof a degenerate library of genes encoding a library of polypeptideswhich each include at least a portion of potential ActRII polypeptidesequences. For instance, a mixture of synthetic oligonucleotides can beenzymatically ligated into gene sequences such that the degenerate setof potential ActRII polypeptide nucleotide sequences are expressible asindividual polypeptides, or alternatively, as a set of larger fusionproteins (e.g., for phage display).

There are many ways by which the library of potential homologs can begenerated from a degenerate oligonucleotide sequence. Chemical synthesisof a degenerate gene sequence can be carried out in an automatic DNAsynthesizer, and the synthetic genes then be ligated into an appropriatevector for expression. The synthesis of degenerate oligonucleotides iswell known in the art (see for example, Narang, S A (1983) Tetrahedron39:3; Itakura et al., (1981) Recombinant DNA, Proc. 3rd ClevelandSympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp 273-289;Itakura et al., (1984) Annu. Rev. Biochem. 53:323; Itakura et al.,(1984) Science 198:1056; Ike et al., (1983) Nucleic Acid Res. 11:477).Such techniques have been employed in the directed evolution of otherproteins (see, for example, Scott et al., (1990) Science 249:386-390;Roberts et al., (1992) PNAS USA 89:2429-2433; Devlin et al., (1990)Science 249: 404-406; Cwirla et al., (1990) PNAS USA 87: 6378-6382; aswell as U.S. Pat. Nos. 5,223,409, 5,198,346, and 5,096,815).

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library. For example, ActRII polypeptide variants (bothagonist and antagonist forms) can be generated and isolated from alibrary by screening using, for example, alanine scanning mutagenesisand the like (Ruf et al., (1994) Biochemistry 33:1565-1572; Wang et al.,(1994) J. Biol. Chem. 269:3095-3099; Balint et al., (1993) Gene137:109-118; Grodberg et al., (1993) Eur. J. Biochem. 218:597-601;Nagashima et al., (1993) J. Biol. Chem. 268:2888-2892; Lowman et al.,(1991) Biochemistry 30:10832-10838; and Cunningham et al., (1989)Science 244:1081-1085), by linker scanning mutagenesis (Gustin et al.,(1993) Virology 193:653-660; Brown et al., (1992) Mol. Cell Biol.12:2644-2652; McKnight et al., (1982) Science 232:316); by saturationmutagenesis (Meyers et al., (1986) Science 232:613); by PCR mutagenesis(Leung et al., (1989) Method Cell Mol Biol 1:11-19); or by randommutagenesis, including chemical mutagenesis, etc. (Miller et al., (1992)A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor,N.Y.; and Greener et al., (1994) Strategies in Mol Biol 7:32-34). Linkerscanning mutagenesis, particularly in a combinatorial setting, is anattractive method for identifying truncated (bioactive) forms of ActRIIpolypeptides. In a specific embodiment, similar methods can be used formaking soluble forms of ActRII polypeptides, which can act as agonistsor antagonists of ActRII functions.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations andtruncations, and, for that matter, for screening cDNA libraries for geneproducts having a certain property. Such techniques will be generallyadaptable for rapid screening of the gene libraries generated by thecombinatorial mutagenesis of ActRII polypeptides. The most widely usedtechniques for screening large gene libraries typically comprisescloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected. Each of theillustrative assays described below are amenable to high through-putanalysis as necessary to screen large numbers of degenerate sequencescreated by combinatorial mutagenesis techniques.

In certain embodiments, the ActRII polypeptides of the present inventioninclude peptidomimetics. As used herein, the term “peptidomimetic”includes chemically modified peptides and peptide-like molecules thatcontain non-naturally occurring amino acids, peptoids, and the like.Peptidomimetics provide various advantages over a peptide, includingenhanced stability when administered to a subject. Methods foridentifying a peptidomimetic are well known in the art and include thescreening of databases that contain libraries of potentialpeptidomimetics. For example, the Cambridge Structural Database containsa collection of greater than 300,000 compounds that have known crystalstructures (Allen et al., Acta Crystallogr. Section B, 35:2331 (1979)).Where no crystal structure of a target molecule is available, astructure can be generated using, for example, the program CONCORD(Rusinko et al., J. Chem. Inf. Comput. Sci. 29:251 (1989)). Anotherdatabase, the Available Chemicals Directory (Molecular Design Limited,Informations Systems; San Leandro Calif.), contains about 100,000compounds that are commercially available and also can be searched toidentify potential peptidomimetics of the ActRII polypeptides.

To illustrate, by employing scanning mutagenesis to map the amino acidresidues of an ActRII polypeptide which are involved in binding toanother protein, peptidomimetic compounds can be generated which mimicthose residues involved in binding. For instance, non-hydrolyzablepeptide analogs of such residues can be generated using benzodiazepine(e.g., see Freidinger et al., in Peptides: Chemistry and Biology, G. R.Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine(e.g., see Huffman et al., in Peptides: Chemistry and Biology, G. R.Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substitutedgamma lactam rings (Garvey et al., in Peptides: Chemistry and Biology,G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988),keto-methylene pseudopeptides (Ewenson et al., (1986) J. Med. Chem.29:295; and Ewenson et al., in Peptides: Structure and Function(Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co.Rockland, Ill., 1985), b-turn dipeptide cores (Nagai et al., (1985)Tetrahedron Lett 26:647; and Sato et al., (1986) J Chem Soc Perkin Trans1:1231), and b-aminoalcohols (Gordon et al., (1985) Biochem Biophys ResCommun 126:419; and Dann et al., (1986) Biochem Biophys Res Commun134:71).

In certain embodiments, the ActRII polypeptides of the invention mayfurther comprise post-translational modifications in addition to anythat are naturally present in the ActRII polypeptides. Suchmodifications include, but are not limited to, acetylation,carboxylation, glycosylation, phosphorylation, lipidation, andacylation. As a result, the modified ActRII polypeptides may containnon-amino acid elements, such as polyethylene glycols, lipids, poly- ormono-saccharide, and phosphates. Effects of such non-amino acid elementson the functionality of a ActRII polypeptide may be tested as describedherein for other ActRII polypeptide variants. When an ActRII polypeptideis produced in cells by cleaving a nascent form of the ActRIIpolypeptide, post-translational processing may also be important forcorrect folding and/or function of the protein. Different cells (such asCHO, HeLa, MDCK, 293, W138, NIH-3T3 or HEK293) have specific cellularmachinery and characteristic mechanisms for such post-translationalactivities and may be chosen to ensure the correct modification andprocessing of the ActRII polypeptides.

In certain aspects, functional variants or modified forms of the ActRIIpolypeptides include fusion proteins having at least a portion of theActRII polypeptides and one or more fusion domains. Well known examplesof such fusion domains include, but are not limited to, polyhistidine,Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A,protein G, an immunoglobulin heavy chain constant region (Fc), maltosebinding protein (MBP), or human serum albumin. A fusion domain may beselected so as to confer a desired property. For example, some fusiondomains are particularly useful for isolation of the fusion proteins byaffinity chromatography. For the purpose of affinity purification,relevant matrices for affinity chromatography, such as glutathione-,amylase-, and nickel- or cobalt-conjugated resins are used. Many of suchmatrices are available in “kit” form, such as the Pharmacia GSTpurification system and the QIAexpress™ system (Qiagen) useful with(HIS₆) fusion partners. As another example, a fusion domain may beselected so as to facilitate detection of the ActRII polypeptides.Examples of such detection domains include the various fluorescentproteins (e.g., GFP) as well as “epitope tags,” which are usually shortpeptide sequences for which a specific antibody is available. Well knownepitope tags for which specific monoclonal antibodies are readilyavailable include FLAG, influenza virus haemagglutinin (HA), and c-myctags. In some cases, the fusion domains have a protease cleavage site,such as for Factor Xa or Thrombin, which allows the relevant protease topartially digest the fusion proteins and thereby liberate therecombinant proteins therefrom. The liberated proteins can then beisolated from the fusion domain by subsequent chromatographicseparation. In certain preferred embodiments, an ActRII polypeptide isfused with a domain that stabilizes the ActRII polypeptide in vivo (a“stabilizer” domain). By “stabilizing” is meant anything that increasesserum half life, regardless of whether this is because of decreaseddestruction, decreased clearance by the kidney, or other pharmacokineticeffect. Fusions with the Fc portion of an immunoglobulin are known toconfer desirable pharmacokinetic properties on a wide range of proteins.Likewise, fusions to human serum albumin can confer desirableproperties. Other types of fusion domains that may be selected includemultimerizing (e.g., dimerizing, tetramerizing) domains and functionaldomains (that confer an additional biological function, such as furtherstimulation of muscle growth).

As a specific example, the present invention provides a fusion proteinas a GDF8 antagonist which comprises an extracellular (e.g.,GDF8-binding) domain fused to an Fc domain (e.g., SEQ ID NO: 13).

THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD(A)VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK(A)VSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN(A)HYTQKSLSLSPGK*

Preferably, the Fc domain has one or more mutations at residues such asAsp-265, lysine 322, and Asn-434 (see FIG. 12). In certain cases, themutant Fc domain having one or more of these mutations (e.g., Asp-265mutation) has reduced ability of binding to the Fcγ receptor relative toa wildtype Fc domain. In other cases, the mutant Fc domain having one ormore of these mutations (e.g., Asn-434 mutation) has increased abilityof binding to the MHC class I-related Fc-receptor (FcRN) relative to awildtype Fc domain.

It is understood that different elements of the fusion proteins may bearranged in any manner that is consistent with the desiredfunctionality. For example, an ActRII polypeptide may be placedC-terminal to a heterologous domain, or, alternatively, a heterologousdomain may be placed C-terminal to an ActRII polypeptide. The ActRIIpolypeptide domain and the heterologous domain need not be adjacent in afusion protein, and additional domains or amino acid sequences may beincluded C- or N-terminal to either domain or between the domains.

In certain embodiments, the ActRII polypeptides of the present inventioncontain one or more modifications that are capable of stabilizing theActRII polypeptides. For example, such modifications enhance the invitro half life of the ActRII polypeptides, enhance circulatory halflife of the ActRII polypeptides or reducing proteolytic degradation ofthe ActRII polypeptides. Such stabilizing modifications include, but arenot limited to, fusion proteins (including, for example, fusion proteinscomprising an ActRII polypeptide and a stabilizer domain), modificationsof a glycosylation site (including, for example, addition of aglycosylation site to an ActRII polypeptide), and modifications ofcarbohydrate moiety (including, for example, removal of carbohydratemoieties from an ActRII polypeptide). In the case of fusion proteins, anActRII polypeptide is fused to a stabilizer domain such as an IgGmolecule (e.g., an Fc domain). As used herein, the term “stabilizerdomain” not only refers to a fusion domain (e.g., Fc) as in the case offusion proteins, but also includes nonproteinaceous modifications suchas a carbohydrate moiety, or nonproteinaceous polymer, such aspolyethylene glycol.

In certain embodiments, the present invention makes available isolatedand/or purified forms of the ActRII polypeptides, which are isolatedfrom, or otherwise substantially free of, other proteins.

In certain embodiments, ActRII polypeptides (unmodified or modified) ofthe invention can be produced by a variety of art-known techniques. Forexample, such ActRII polypeptides can be synthesized using standardprotein chemistry techniques such as those described in Bodansky, M.Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) andGrant G. A. (ed.), Synthetic Peptides: A User's Guide, W.H. Freeman andCompany, New York (1992). In addition, automated peptide synthesizersare commercially available (e.g., Advanced ChemTech Model 396;Milligen/Biosearch 9600). Alternatively, the ActRII polypeptides,fragments or variants thereof may be recombinantly produced usingvarious expression systems (e.g., E. coli, Chinese Hamster Ovary cells,COS cells, baculovirus) as is well known in the art (also see below). Ina further embodiment, the modified or unmodified ActRII polypeptides maybe produced by digestion of naturally occurring or recombinantlyproduced full-length ActRII polypeptides by using, for example, aprotease, e.g., trypsin, thermolysin, chymotrypsin, pepsin, or pairedbasic amino acid converting enzyme (PACE). Computer analysis (using acommercially available software, e.g., MacVector, Omega, PCGene,Molecular Simulation, Inc.) can be used to identify proteolytic cleavagesites. Alternatively, such ActRII polypeptides may be produced fromnaturally occurring or recombinantly produced full-length ActRIIpolypeptides such as standard techniques known in the art, such as bychemical cleavage (e.g., cyanogen bromide, hydroxylamine).

3. Nucleic Acids Encoding ActRII Polypeptides

In certain aspects, the invention provides isolated and/or recombinantnucleic acids encoding any of the ActRII polypeptides (e.g., solubleActRII polypeptides), including fragments, functional variants andfusion proteins disclosed herein. For example, SEQ ID NOs: 7-8 encodenaturally occurring ActRII precursor polypeptides, while SEQ ID NOs: 5-6encode soluble ActRII polypeptides. The subject nucleic acids may besingle-stranded or double stranded. Such nucleic acids may be DNA or RNAmolecules. These nucleic acids are may be used, for example, in methodsfor making ActRII polypeptides or as direct therapeutic agents (e.g., ina gene therapy approach).

In certain aspects, the subject nucleic acids encoding ActRIIpolypeptides are further understood to include nucleic acids that arevariants of SEQ ID NO: 7 or 8. Variant nucleotide sequences includesequences that differ by one or more nucleotide substitutions, additionsor deletions, such as allelic variants; and will, therefore, includecoding sequences that differ from the nucleotide sequence of the codingsequence designated in SEQ ID NO: 7 or 8.

In certain embodiments, the invention provides isolated or recombinantnucleic acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%,99% or 100% identical to SEQ ID NO: 5 or 6. One of ordinary skill in theart will appreciate that nucleic acid sequences complementary to SEQ IDNO: 5 or 6, and variants of SEQ ID NO: 5 or 6 are also within the scopeof this invention. In further embodiments, the nucleic acid sequences ofthe invention can be isolated, recombinant, and/or fused with aheterologous nucleotide sequence, or in a DNA library.

In other embodiments, nucleic acids of the invention also includenucleotide sequences that hybridize under highly stringent conditions tothe nucleotide sequence designated in SEQ ID NO: 5 or 6, complementsequence of SEQ ID NO: 5 or 6, or fragments thereof. As discussed above,one of ordinary skill in the art will understand readily thatappropriate stringency conditions which promote DNA hybridization can bevaried. One of ordinary skill in the art will understand readily thatappropriate stringency conditions which promote DNA hybridization can bevaried. For example, one could perform the hybridization at 6.0× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by a wash of2.0×SSC at 50° C. For example, the salt concentration in the wash stepcan be selected from a low stringency of about 2.0×SSC at 50° C. to ahigh stringency of about 0.2×SSC at 50° C. In addition, the temperaturein the wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or temperature or saltconcentration may be held constant while the other variable is changed.In one embodiment, the invention provides nucleic acids which hybridizeunder low stringency conditions of 6×SSC at room temperature followed bya wash at 2×SSC at room temperature.

Isolated nucleic acids which differ from the nucleic acids as set forthin SEQ ID NOs: 5-6 due to degeneracy in the genetic code are also withinthe scope of the invention. For example, a number of amino acids aredesignated by more than one triplet. Codons that specify the same aminoacid, or synonyms (for example, CAU and CAC are synonyms for histidine)may result in “silent” mutations which do not affect the amino acidsequence of the protein. However, it is expected that DNA sequencepolymorphisms that do lead to changes in the amino acid sequences of thesubject proteins will exist among mammalian cells. One skilled in theart will appreciate that these variations in one or more nucleotides (upto about 3-5% of the nucleotides) of the nucleic acids encoding aparticular protein may exist among individuals of a given species due tonatural allelic variation. Any and all such nucleotide variations andresulting amino acid polymorphisms are within the scope of thisinvention.

In certain embodiments, the recombinant nucleic acids of the inventionmay be operably linked to one or more regulatory nucleotide sequences inan expression construct. Regulatory nucleotide sequences will generallybe appropriate to the host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the invention. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome. In a preferred embodiment, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selectable marker genes are well known in the art and willvary with the host cell used.

In certain aspects of the invention, the subject nucleic acid isprovided in an expression vector comprising a nucleotide sequenceencoding an ActRII polypeptide and operably linked to at least oneregulatory sequence. Regulatory sequences are art-recognized and areselected to direct expression of the ActRII polypeptide. Accordingly,the term regulatory sequence includes promoters, enhancers, and otherexpression control elements. Exemplary regulatory sequences aredescribed in Goeddel; Gene Expression Technology: Methods in Enzymology,Academic Press, San Diego, Calif. (1990). For instance, any of a widevariety of expression control sequences that control the expression of aDNA sequence when operatively linked to it may be used in these vectorsto express DNA sequences encoding an ActRII polypeptide. Such usefulexpression control sequences, include, for example, the early and latepromoters of SV40, tet promoter, adenovirus or cytomegalovirus immediateearly promoter, RSV promoters, the lac system, the trp system, the TACor TRC system, T7 promoter whose expression is directed by T7 RNApolymerase, the major operator and promoter regions of phage lambda, thecontrol regions for fd coat protein, the promoter for 3-phosphoglyceratekinase or other glycolytic enzymes, the promoters of acid phosphatase,e.g., Pho5, the promoters of the yeast α-mating factors, the polyhedronpromoter of the baculovirus system and other sequences known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof. It should be understood thatthe design of the expression vector may depend on such factors as thechoice of the host cell to be transformed and/or the type of proteindesired to be expressed. Moreover, the vector's copy number, the abilityto control that copy number and the expression of any other proteinencoded by the vector, such as antibiotic markers, should also beconsidered.

A recombinant nucleic acid of the invention can be produced by ligatingthe cloned gene, or a portion thereof, into a vector suitable forexpression in either prokaryotic cells, eukaryotic cells (yeast, avian,insect or mammalian), or both. Expression vehicles for production of arecombinant ActRII polypeptide include plasmids and other vectors. Forinstance, suitable vectors include plasmids of the types: pBR322-derivedplasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derivedplasmids and pUC-derived plasmids for expression in prokaryotic cells,such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription units that are expressed in eukaryotic cells.The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found below inthe description of gene therapy delivery systems. The various methodsemployed in the preparation of the plasmids and in transformation ofhost organisms are well known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures, see Molecular Cloning A Laboratory Manual, 2ndEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press, 1989) Chapters 16 and 17. In some instances, it may bedesirable to express the recombinant polypeptides by the use of abaculovirus expression system. Examples of such baculovirus expressionsystems include pVL-derived vectors (such as pVL1392, pVL1393 andpVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derivedvectors (such as the β-gal containing pBlueBac III).

In a preferred embodiment, a vector will be designed for production ofthe subject ActRII polypeptides in CHO cells, such as a Pcmv-Scriptvector (Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen,Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison, Wis.). As willbe apparent, the subject gene constructs can be used to cause expressionof the subject ActRII polypeptides in cells propagated in culture, e.g.,to produce proteins, including fusion proteins or variant proteins, forpurification.

This invention also pertains to a host cell transfected with arecombinant gene including a coding sequence (e.g., SEQ ID NO: 7 or 8)for one or more of the subject ActRII polypeptides. The host cell may beany prokaryotic or eukaryotic cell. For example, an ActRII polypeptideof the invention may be expressed in bacterial cells such as E. coli,insect cells (e.g., using a baculovirus expression system), yeast, ormammalian cells. Other suitable host cells are known to those skilled inthe art.

Accordingly, the present invention further pertains to methods ofproducing the subject ActRII polypeptides. For example, a host celltransfected with an expression vector encoding an ActRII polypeptide canbe cultured under appropriate conditions to allow expression of theActRII polypeptide to occur. The ActRII polypeptide may be secreted andisolated from a mixture of cells and medium containing the ActRIIpolypeptide. Alternatively, the ActRII polypeptide may be retainedcytoplasmically or in a membrane fraction and the cells harvested, lysedand the protein isolated. A cell culture includes host cells, media andother byproducts. Suitable media for cell culture are well known in theart. The subject ActRII polypeptides can be isolated from cell culturemedium, host cells, or both, using techniques known in the art forpurifying proteins, including ion-exchange chromatography, gelfiltration chromatography, ultrafiltration, electrophoresis, andimmunoaffinity purification with antibodies specific for particularepitopes of the ActRII polypeptides. In a preferred embodiment, theActRII polypeptide is a fusion protein containing a domain whichfacilitates its purification.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant ActRIIpolypeptide, can allow purification of the expressed fusion protein byaffinity chromatography using a Ni²⁺ metal resin. The purificationleader sequence can then be subsequently removed by treatment withenterokinase to provide the purified ActRII polypeptide (e.g., seeHochuli et al., (1987) J. Chromatography 411:177; and Janknecht et al.,PNAS USA 88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons: 1992).

4. Antibodies

Another aspect of the invention pertains to antibodies. An antibody thatis specifically reactive with an ActRII polypeptide (e.g., a solubleActRII polypeptide) and which binds competitively with the ActRIIpolypeptide may be used as an antagonist of ActRII polypeptideactivities. For example, by using immunogens derived from an ActRIIpolypeptide, anti-protein/anti-peptide antisera or monoclonal antibodiescan be made by standard protocols (see, for example, Antibodies: ALaboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press:1988)). A mammal, such as a mouse, a hamster or rabbit can be immunizedwith an immunogenic form of the ActRII polypeptide, an antigenicfragment which is capable of eliciting an antibody response, or a fusionprotein. Techniques for conferring immunogenicity on a protein orpeptide include conjugation to carriers or other techniques well knownin the art. An immunogenic portion of an ActRII polypeptide can beadministered in the presence of adjuvant. The progress of immunizationcan be monitored by detection of antibody titers in plasma or serum.Standard ELISA or other immunoassays can be used with the immunogen asantigen to assess the levels of antibodies.

Following immunization of an animal with an antigenic preparation of anActRII polypeptide, antisera can be obtained and, if desired, polyclonalantibodies can be isolated from the serum. To produce monoclonalantibodies, antibody-producing cells (lymphocytes) can be harvested froman immunized animal and fused by standard somatic cell fusion procedureswith immortalizing cells such as myeloma cells to yield hybridoma cells.Such techniques are well known in the art, and include, for example, thehybridoma technique (originally developed by Kohler and Milstein, (1975)Nature, 256: 495-497), the human B cell hybridoma technique (Kozbar etal., (1983) Immunology Today, 4: 72), and the EBV-hybridoma technique toproduce human monoclonal antibodies (Cole et al., (1985) MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96). Hybridomacells can be screened immunochemically for production of antibodiesspecifically reactive with an ActRII polypeptide and monoclonalantibodies isolated from a culture comprising such hybridoma cells.

The term “antibody” as used herein is intended to include fragmentsthereof which are also specifically reactive with a subject ActRIIpolypeptide. Antibodies can be fragmented using conventional techniquesand the fragments screened for utility in the same manner as describedabove for whole antibodies. For example, F(ab)₂ fragments can begenerated by treating antibody with pepsin. The resulting F(ab)₂fragment can be treated to reduce disulfide bridges to produce Fabfragments. The antibody of the present invention is further intended toinclude bispecific, single-chain, and chimeric and humanized moleculeshaving affinity for an ActRII polypeptide conferred by at least one CDRregion of the antibody. In preferred embodiments, the antibody furthercomprises a label attached thereto and able to be detected (e.g., thelabel can be a radioisotope, fluorescent compound, enzyme or enzymeco-factor).

In certain preferred embodiments, an antibody of the invention is amonoclonal antibody, and in certain embodiments, the invention makesavailable methods for generating novel antibodies. For example, a methodfor generating a monoclonal antibody that binds specifically to anActRII polypeptide may comprise administering to a mouse an amount of animmunogenic composition comprising the ActRII polypeptide effective tostimulate a detectable immune response, obtaining antibody-producingcells (e.g., cells from the spleen) from the mouse and fusing theantibody-producing cells with myeloma cells to obtain antibody-producinghybridomas, and testing the antibody-producing hybridomas to identify ahybridoma that produces a monocolonal antibody that binds specificallyto the ActRII polypeptide. Once obtained, a hybridoma can be propagatedin a cell culture, optionally in culture conditions where thehybridoma-derived cells produce the monoclonal antibody that bindsspecifically to the ActRII polypeptide. The monoclonal antibody may bepurified from the cell culture.

The adjective “specifically reactive with” as used in reference to anantibody is intended to mean, as is generally understood in the art,that the antibody is sufficiently selective between the antigen ofinterest (e.g., an ActRII polypeptide) and other antigens that are notof interest that the antibody is useful for, at minimum, detecting thepresence of the antigen of interest in a particular type of biologicalsample. In certain methods employing the antibody, such as therapeuticapplications, a higher degree of specificity in binding may bedesirable. Monoclonal antibodies generally have a greater tendency (ascompared to polyclonal antibodies) to discriminate effectively betweenthe desired antigens and cross-reacting polypeptides. One characteristicthat influences the specificity of an antibody:antigen interaction isthe affinity of the antibody for the antigen. Although the desiredspecificity may be reached with a range of different affinities,generally preferred antibodies will have an affinity (a dissociationconstant) of about 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ or less.

In addition, the techniques used to screen antibodies in order toidentify a desirable antibody may influence the properties of theantibody obtained. For example, if an antibody is to be used for bindingan antigen in solution, it may be desirable to test solution binding. Avariety of different techniques are available for testing interactionbetween antibodies and antigens to identify particularly desirableantibodies. Such techniques include ELISAs, surface plasmon resonancebinding assays (e.g., the Biacore binding assay, Bia-core AB, Uppsala,Sweden), sandwich assays (e.g., the paramagnetic bead system of IGENInternational, Inc., Gaithersburg, Md.), western blots,immunoprecipitation assays, and immunohistochemistry.

In certain aspects, the disclosure provides antibodies that bind to asoluble ActRII polypeptide. Such antibodies may be generated much asdescribed above, using a soluble ActRII polypeptide or fragment thereofas an antigen. Antibodies of this type can be used, e.g., to detectActRII polypeptides in biological samples and/or to monitor solubleActRII polypeptide levels in an individual. In certain cases, anantibody that specifically binds to a soluble ActRII polypeptide can beused to modulate activity of an ActRII polypeptide and/or an ActRIIligand, thereby regulating (promoting or inhibiting) growth of tissues,such as bone, cartilage, muscle, fat, and neurons.

5. Screening Assays

In certain aspects, the present invention relates to the use of thesubject ActRII polypeptides (e.g., soluble ActRII polypeptides) toidentify compounds (agents) which are agonist or antagonists of theActRII polypeptides. Compounds identified through this screening can betested in tissues such as bone, cartilage, muscle, fat, and/or neurons,to assess their ability to modulate tissue growth in vitro. Optionally,these compounds can further be tested in animal models to assess theirability to modulate tissue growth in vivo.

There are numerous approaches to screening for therapeutic agents formodulating tissue growth by targeting the ActRII polypeptides. Incertain embodiments, high-throughput screening of compounds can becarried out to identify agents that perturb ActRII-mediated effects ongrowth of bone, cartilage, muscle, fat, and/or neuron. In certainembodiments, the assay is carried out to screen and identify compoundsthat specifically inhibit or reduce binding of an ActRII polypeptide toits binding partner, such as an ActRII ligand (e.g., activin, Nodal,GDF8, GDF11 or BMP7). Alternatively, the assay can be used to identifycompounds that enhance binding of an ActRII polypeptide to its bindingprotein such as an ActRII ligand. In a further embodiment, the compoundscan be identified by their ability to interact with an ActRIIpolypeptide.

A variety of assay formats will suffice and, in light of the presentdisclosure, those not expressly described herein will nevertheless becomprehended by one of ordinary skill in the art. As described herein,the test compounds (agents) of the invention may be created by anycombinatorial chemical method. Alternatively, the subject compounds maybe naturally occurring biomolecules synthesized in vivo or in vitro.Compounds (agents) to be tested for their ability to act as modulatorsof tissue growth can be produced, for example, by bacteria, yeast,plants or other organisms (e.g., natural products), produced chemically(e.g., small molecules, including peptidomimetics), or producedrecombinantly. Test compounds contemplated by the present inventioninclude non-peptidyl organic molecules, peptides, polypeptides,peptidomimetics, sugars, hormones, and nucleic acid molecules. In aspecific embodiment, the test agent is a small organic molecule having amolecular weight of less than about 2,000 daltons.

The test compounds of the invention can be provided as single, discreteentities, or provided in libraries of greater complexity, such as madeby combinatorial chemistry. These libraries can comprise, for example,alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers andother classes of organic compounds. Presentation of test compounds tothe test system can be in either an isolated form or as mixtures ofcompounds, especially in initial screening steps. Optionally, thecompounds may be optionally derivatized with other compounds and havederivatizing groups that facilitate isolation of the compounds.Non-limiting examples of derivatizing groups include biotin,fluorescein, digoxygenin, green fluorescent protein, isotopes,polyhistidine, magnetic beads, glutathione S transferase (GST),photoactivatible crosslinkers or any combinations thereof.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity or bioavailability of the test compound canbe generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity between an ActRIIpolypeptide and its binding protein (e.g., an ActRII ligand).

Merely to illustrate, in an exemplary screening assay of the presentinvention, the compound of interest is contacted with an isolated andpurified ActRII polypeptide which is ordinarily capable of binding to anActRII ligand, as appropriate for the intention of the assay. To themixture of the compound and ActRII polypeptide is then added acomposition containing an ActRII ligand. Detection and quantification ofActRII/ActRII ligand complexes provides a means for determining thecompound's efficacy at inhibiting (or potentiating) complex formationbetween the ActRII polypeptide and its binding protein. The efficacy ofthe compound can be assessed by generating dose response curves fromdata obtained using various concentrations of the test compound.Moreover, a control assay can also be performed to provide a baselinefor comparison. For example, in a control assay, isolated and purifiedActRII ligand is added to a composition containing the ActRIIpolypeptide, and the formation of ActRII/ActRII ligand complex isquantitated in the absence of the test compound. It will be understoodthat, in general, the order in which the reactants may be admixed can bevaried, and can be admixed simultaneously. Moreover, in place ofpurified proteins, cellular extracts and lysates may be used to render asuitable cell-free assay system.

Complex formation between the ActRII polypeptide and its binding proteinmay be detected by a variety of techniques. For instance, modulation ofthe formation of complexes can be quantitated using, for example,detectably labeled proteins such as radiolabeled (e.g., ³²P, ³⁵S, ¹⁴C or³H), fluorescently labeled (e.g., FITC), or enzymatically labeled ActRIIpolypeptide or its binding protein, by immunoassay, or bychromatographic detection.

In certain embodiments, the present invention contemplates the use offluorescence polarization assays and fluorescence resonance energytransfer (FRET) assays in measuring, either directly or indirectly, thedegree of interaction between an ActRII polypeptide and its bindingprotein. Further, other modes of detection, such as those based onoptical waveguides (PCT Publication WO 96/26432 and U.S. Pat. No.5,677,196), surface plasmon resonance (SPR), surface charge sensors, andsurface force sensors, are compatible with many embodiments of theinvention.

Moreover, the present invention contemplates the use of an interactiontrap assay, also known as the “two hybrid assay,” for identifying agentsthat disrupt or potentiate interaction between an ActRII polypeptide andits binding protein. See for example, U.S. Pat. No. 5,283,317; Zervos etal. (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; andIwabuchi et al. (1993) Oncogene 8:1693-1696). In a specific embodiment,the present invention contemplates the use of reverse two hybrid systemsto identify compounds (e.g., small molecules or peptides) thatdissociate interactions between an ActRII polypeptide and its bindingprotein. See for example, Vidal and Legrain, (1999) Nucleic Acids Res27:919-29; Vidal and Legrain, (1999) Trends Biotechnol 17:374-81; andU.S. Pat. Nos. 5,525,490; 5,955,280; and 5,965,368.

In certain embodiments, the subject compounds are identified by theirability to interact with an ActRII polypeptide of the invention. Theinteraction between the compound and the ActRII polypeptide may becovalent or non-covalent. For example, such interaction can beidentified at the protein level using in vitro biochemical methods,including photo-crosslinking, radiolabeled ligand binding, and affinitychromatography (Jakoby W B et al., 1974, Methods in Enzymology 46: 1).In certain cases, the compounds may be screened in a mechanism basedassay, such as an assay to detect compounds which bind to an ActRIIpolypeptide. This may include a solid phase or fluid phase bindingevent. Alternatively, the gene encoding an ActRII polypeptide can betransfected with a reporter system (e.g., β-galactosidase, luciferase,or green fluorescent protein) into a cell and screened against thelibrary preferably by a high throughput screening or with individualmembers of the library. Other mechanism based binding assays may beused, for example, binding assays which detect changes in free energy.Binding assays can be performed with the target fixed to a well, bead orchip or captured by an immobilized antibody or resolved by capillaryelectrophoresis. The bound compounds may be detected usually usingcolorimetric or fluorescence or surface plasmon resonance.

In certain aspects, the present invention provides methods and agentsfor stimulating muscle growth and increasing muscle mass, for example,by antagonizing functions of an ActRII polypeptide and/or an ActRIIligand. Therefore, any compound identified can be tested in whole cellsor tissues, in vitro or in vivo, to confirm their ability to modulatemuscle growth. Various methods known in the art can be utilized for thispurpose. For example, methods of the invention are performed such thatthe signal transduction through an ActRII protein activated by bindingto an ActRII ligand (e.g., GDF8) has been reduced or inhibited. It willbe recognized that the growth of muscle tissue in the organism wouldresult in an increased muscle mass in the organism as compared to themuscle mass of a corresponding organism (or population of organisms) inwhich the signal transduction through an ActRII protein had not been soeffected.

For example, the effect of the ActRII polypeptides or test compounds onmuscle cell growth/proliferation can be determined by measuring geneexpression of Pax-3 and Myf-5 which are associated with proliferation ofmyogenic cells, and gene expression of MyoD which is associated withmuscle differentiation (e.g., Amthor et al., Dev Biol. 2002,251:241-57). It is known that GDF8 down-regulates gene expression ofPax-3 and Myf-5, and prevents gene expression of MyoD. The ActRIIpolypeptides or test compounds are expected to antagonize this activityof GDF8. Another example of cell-based assays includes measuring theproliferation of myoblasts such as C(2)C(12) myoblasts in the presenceof the ActRII polypeptides or test compounds (e.g., Thomas et al., JBiol. Chem. 2000, 275:40235-43).

The present invention also contemplates in vivo assays to measure musclemass and strength. For example, Whittemore et al. (Biochem Biophys ResCommun. 2003, 300:965-71) discloses a method of measuring increasedskeletal muscle mass and increased grip strength in mice. Optionally,this method can be used to determine therapeutic effects of testcompounds (e.g., ActRII polypeptides) on muscle diseases or conditions,for example those diseases for which muscle mass is limiting.

In certain aspects, the present invention provides methods and agentsfor modulating (stimulating or inhibiting) bone formation and increasingbone mass. Therefore, any compound identified can be tested in wholecells or tissues, in vitro or in vivo, to confirm their ability tomodulate bone or cartilage growth. Various methods known in the art canbe utilized for this purpose.

For example, the effect of the ActRII polypeptides or test compounds onbone or cartilage growth can be determined by measuring induction ofMsx2 or differentiation of osteoprogenitor cells into osteoblasts incell based assays (see, e.g., Daluiski et al., Nat Genet. 2001,27(1):84-8; Hino et al., Front Biosci. 2004, 9:1520-9). Another exampleof cell-based assays includes analyzing the osteogenic activity of thesubject ActRII polypeptides and test compounds in mesenchymal progenitorand osteoblastic cells. To illustrate, recombinant adenovirusesexpressing an ActRII polypeptide were constructed to infect pluripotentmesenchymal progenitor C3H10T1/2 cells, preosteoblastic C2C12 cells, andosteoblastic TE-85 cells. Osteogenic activity is then determined bymeasuring the induction of alkaline phosphatase, osteocalcin, and matrixmineralization (see, e.g., Cheng et al., J bone Joint Surg Am. 2003,85-A(8):1544-52).

The present invention also contemplates in vivo assays to measure boneor cartilage growth. For example, Namkung-Matthai et al., Bone, 28:80-86(2001) discloses a rat osteoporotic model in which bone repair duringthe early period after fracture is studied. Kubo et al., SteroidBiochemistry & Molecular Biology, 68:197-202 (1999) also discloses a ratosteoporotic model in which bone repair during the late period afterfracture is studied. These references are incorporated by referenceherein in their entirety for their disclosure of rat model for study onosteoporotic bone fracture. In certain aspects, the present inventionmakes use of fracture healing assays that are known in the art. Theseassays include fracture technique, histological analysis, andbiomechanical analysis, which are described in, for example, U.S. Pat.No. 6,521,750, which is incorporated by reference in its entirety forits disclosure of experimental protocols for causing as well asmeasuring the extent of fractures, and the repair process.

In certain aspects, the present invention provides methods and agentsfor controlling weight gain and obesity. At the cellular level,adipocyte proliferation and differentiation is critical in thedevelopment of obesity, which leads to the generation of additional fatcells (adipocytes). Therefore, any compound identified can be tested inwhole cells or tissues, in vitro or in vivo, to confirm their ability tomodulate adipogenesis by measuring adipocyte proliferation ordifferentiation. Various methods known in the art can be utilized forthis purpose. For example, the effect of an ActRII polypeptide (e.g., asoluble ActRII polypeptide) or test compounds on adipogenesis can bedetermined by measuring differentiation of 3T3-L1 preadipocytes tomature adipocytes in cell based assays, such as, by observing theaccumulation of triacylglycerol in Oil Red O staining vesicles and bythe appearance of certain adipocyte markers such as FABP (aP2/422) andPPARγ2. See, for example, Reusch et al., 2000, Mol Cell Biol.20:1008-20; Deng et al., 2000, Endocrinology. 141:2370-6; Bell et al.,2000, Obes Res. 8:249-54. Another example of cell-based assays includesanalyzing the role of ActRII. polypeptides and test compounds inproliferation of adipocytes or adipocyte precursor cells (e.g., 3T3-L1cells), such as, by monitoring bromodeoxyuridine (BrdU)-positive cells.See, for example, Pico et al., 1998, Mol Cell Biochem. 189:1-7; Masunoet al., 2003, Toxicol Sci. 75:314-20.

It is understood that the screening assays of the present inventionapply to not only the subject ActRII polypeptides and variants of theActRII polypeptides, but also any test compounds including agonists andantagonist of the ActRII polypeptides. Further, these screening assaysare useful for drug target verification and quality control purposes.

6. Exemplary Therapeutic Uses

In certain embodiments, compositions (e.g., ActRII polypeptides) of thepresent invention can be used for treating or preventing a disease orcondition that is associated with abnormal activity of an ActRIIpolypeptide and/or an ActRII ligand (e.g., GDF8). These diseases,disorders or conditions are generally referred to herein as“ActRII-associated conditions.” In certain embodiments, the presentinvention provides methods of treating or preventing an individual inneed thereof through administering to the individual a therapeuticallyeffective amount of an ActRII polypeptide as described above. Thesemethods are particularly aimed at therapeutic and prophylactictreatments of animals, and more particularly, humans.

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample. The term “treating” as used hereinincludes prophylaxis of the named condition or amelioration orelimination of the condition once it has been established.

ActRII/ActRII ligand complexes play essential roles in tissue growth aswell as early developmental processes such as the correct formation ofvarious structures or in one or more post-developmental capacitiesincluding sexual development, pituitary hormone production, and creationof bone and cartilage. Thus, ActRII-associated conditions includeabnormal tissue growth and developmental defects. In addition,ActRII-associated conditions include, but are not limited to, disordersof cell growth and differentiation such as inflammation, allergy,autoimmune diseases, infectious diseases, and tumors.

Exemplary ActRII-associated conditions include neuromuscular disorders(e.g., muscular dystrophy and muscle atrophy), congestive obstructivepulmonary disease, muscle wasting syndrome, sarcopenia, cachexia,adipose tissue disorders (e.g., obesity), type 2 diabetes, and bonedegenerative disease (e.g., osteoporosis). Other exemplaryActRII-associated conditions include musculodegenerative andneuromuscular disorders, tissue repair (e.g., wound healing),neurodegenerative diseases (e.g., amyotrophic lateral sclerosis),immunologic disorders (e.g., disorders related to abnormal proliferationor function of lymphocytes), and obesity or disorders related toabnormal proliferation of adipocytes.

In certain embodiments, compositions (e.g., soluble ActRII polypeptides)of the invention are used as part of a treatment for a musculardystrophy. The term “muscular dystrophy” refers to a group ofdegenerative muscle diseases characterized by gradual weakening anddeterioration of skeletal muscles and sometimes the heart andrespiratory muscles. Muscular dystrophies are genetic disorderscharacterized by progressive muscle wasting and weakness that begin withmicroscopic changes in the muscle. As muscles degenerate over time, theperson's muscle strength declines. Exemplary muscular dystrophies thatcan be treated with a regimen including the subject ActRII polypeptidesinclude: Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy(BMD), Emery-Dreifuss Muscular Dystrophy (EDMD), Limb-Girdle MuscularDystrophy (LGMD), Facioscapulohumeral Muscular Dystrophy (FSH or FSHD)(also known as Landouzy-Dejerine), Myotonic Dystrophy-(MMD) (also knownas Steinert's Disease), Oculopharyngeal Muscular Dystrophy (OPMD),Distal Muscular Dystrophy (DD), Congenital Muscular Dystrophy (CMD).

Duchenne Muscular Dystrophy (DMD) was first described by the Frenchneurologist Guillaume Benjamin Amand Duchenne in the 1860s. BeckerMuscular Dystrophy (BMD) is named after the German doctor Peter EmilBecker, who first described this variant of DMD in the 1950s. DMD is oneof the most frequent inherited diseases in males, affecting one in 3,500boys. DMD occurs when the dystrophin gene, located on the short arm ofthe X chromosome, is broken. Since males only carry one copy of the Xchromosome, they only have one copy of the dystrophin gene. Without thedystrophin protein, muscle is easily damaged during cycles ofcontraction and relaxation. While early in the disease musclecompensates by regeneration, later on muscle progenitor cells cannotkeep up with the ongoing damage and healthy muscle is replaced bynon-functional fibro-fatty tissue.

BMD results from different mutations in the dystrophin gene. BMDpatients have some dystrophin, but it is either insufficient in quantityor poor in quality. Having some dystrophin protects the muscles of thosewith BMD from degenerating as badly or as quickly as those of peoplewith DMD.

For example, recent researches demonstrate that blocking or eliminatingfunction of GDF8 (an ActRII ligand) in vivo can effectively treat atleast certain symptoms in DMD and BMD patients (Bogdanovich et al.,supra; Wagner et al., supra). Thus, the subject ActRII polypeptides mayact as GDF8 inhibitors (antagonists), and constitute an alternativemeans of blocking the functions of GDF8 and/or ActRII in vivo in DMD andBMD patients.

Similarly, the subject ActRII polypeptides provide an effective means toincrease muscle mass in other disease conditions that are in need ofmuscle growth. For example, Gonzalez-Cadavid et al. (supra) reportedthat that GDF8 expression correlates inversely with fat-free mass inhumans and that increased expression of the GDF8 gene is associated withweight loss in men with AIDS wasting syndrome. By inhibiting thefunction of GDF8 in AIDS patients, at least certain symptoms of AIDS maybe alleviated, if not completely eliminated, thus significantlyimproving quality of life in AIDS patients.

Since loss of GDF8 (an ActRII ligand) function is also associated withfat loss without diminution of nutrient intake (Zimmers et al., supra;McPherron and Lee, supra), the subject ActRII polypeptides may furtherbe used as a therapeutic agent for slowing or preventing the developmentof obesity and type II diabetes.

The cancer anorexia-cachexia syndrome is among the most debilitating andlife-threatening aspects of cancer. Progressive weight loss in canceranorexia-cachexia syndrome is a common feature of many types of cancerand is responsible not only for a poor quality of life and poor responseto chemotherapy, but also a shorter survival time than is found inpatients with comparable tumors without weight loss. Associated withanorexia, fat and muscle tissue wasting, psychological distress, and alower quality of life, cachexia arises from a complex interactionbetween the cancer and the host. It is one of the most common causes ofdeath among cancer patients and is present in 80% at death. It is acomplex example of metabolic chaos effecting protein, carbohydrate, andfat metabolism. Tumors produce both direct and indirect abnormalities,resulting in anorexia and weight loss. Currently, there is no treatmentto control or reverse the process. Cancer anorexia-cachexia syndromeaffects cytokine production, release of lipid-mobilizing andproteolysis-inducing factors, and alterations in intermediarymetabolism. Although anorexia is common, a decreased food intake aloneis unable to account for the changes in body composition seen in cancerpatients, and increasing nutrient intake is unable to reverse thewasting syndrome. Cachexia should be suspected in patients with cancerif an involuntary weight loss of greater than five percent of premorbidweight occurs within a six-month period.

Since systemic overexpression of GDF8 in adult mice was found to induceprofound muscle and fat loss analogous to that seen in human cachexiasyndromes (Zimmers et al., supra), the subject ActRII polypeptides aspharmaceutical compositions can be beneficially used to prevent, treat,or alleviate the symptoms of the cachexia syndrome, where muscle growthis desired.

In other embodiments, the present invention provides methods of inducingbone and/or cartilage formation, preventing bone loss, increasing bonemineralization or preventing the demineralization of bone. For example,the subject ActRII polypeptides and compounds identified in the presentinvention have application in treating osteoporosis and the healing ofbone fractures and cartilage defects in humans and other animals. ActRIIpolypeptides may be useful in patients that are diagnosed withsubclinical low bone density, as a protective measure against thedevelopment of osteoporosis.

In one specific embodiment, methods and compositions of the presentinvention may find medical utility in the healing of bone fractures andcartilage defects in humans and other animals. The subject methods andcompositions may also have prophylactic use in closed as well as openfracture reduction and also in the improved fixation of artificialjoints. De novo bone formation induced by an osteogenic agentcontributes to the repair of congenital, trauma-induced, or oncologicresection induced craniofacial defects, and also is useful in cosmeticplastic surgery. Further, methods and compositions of the invention maybe used in the treatment of periodontal disease, and in other toothrepair processes. In certain cases, the subject ActRII polypeptides mayprovide an environment to attract bone-forming cells, stimulate growthof bone-forming cells or induce differentiation of progenitors ofbone-forming cells. ActRII polypeptides of the invention may also beuseful in the treatment of osteoporosis. Further, ActRII polypeptidesmay be used in cartilage defect repair and prevention/reversal ofosteoarthritis.

In another specific embodiment, the invention provides a therapeuticmethod and composition for repairing fractures and other conditionsrelated to cartilage and/or bone defects or periodontal diseases. Theinvention further provides therapeutic methods and compositions forwound healing and tissue repair. The types of wounds include, but arenot limited to, burns, incisions and ulcers. See e.g., PCT PublicationNo. WO84/01106. Such compositions comprise a therapeutically effectiveamount of at least one of the ActRII polypeptides of the invention inadmixture with a pharmaceutically acceptable vehicle, carrier or matrix.

In another specific embodiment, methods and compositions of theinvention can be applied to conditions causing bone loss such asosteoporosis, hyperparathyroidism, Cushing's disease, thyrotoxicosis,chronic diarrheal state or malabsorption, renal tubular acidosis, oranorexia nervosa. Many people know that being female, having a low bodyweight, and leading a sedentary lifestyle are risk factors forosteoporosis (loss of bone mineral density, leading to fracture risk).However, osteoporosis can also result from the long-term use of certainmedications. Osteoporosis resulting from drugs or another medicalcondition is known as secondary osteoporosis. In a condition known asCushing's disease, the excess amount of cortisol produced by the bodyresults in osteoporosis and fractures. The most common medicationsassociated with secondary osteoporosis are the corticosteroids, a classof drugs that act like cortisol, a hormone produced naturally by theadrenal glands. Although adequate levels of thyroid hormones (which areproduced by the thyroid gland) are needed for the development of theskeleton, excess thyroid hormone can decrease bone mass over time.Antacids that contain aluminum can lead to bone loss when taken in highdoses by people with kidney problems, particularly those undergoingdialysis. Other medications that can cause secondary osteoporosisinclude phenyloin (Dilantin) and barbiturates that are used to preventseizures; methotrexate (Rheumatrex, Immunex, Folex PFS), a drug for someforms of arthritis, cancer, and immune disorders; cyclosporine(Sandimmune, Neoral), a drug used to treat some autoimmune diseases andto suppress the immune system in organ transplant patients; luteinizinghormone-releasing hormone agonists (Lupron, Zoladex), used to treatprostate cancer and endometriosis; heparin (Calciparine, Liquaemin), ananticlotting medication; and cholestyramine (Questran) and colestipol(Colestid), used to treat high cholesterol. Gum disease causes bone lossbecause these harmful bacteria in our mouths force our bodies to defendagainst them. The bacteria produce toxins and enzymes under thegum-line, causing a chronic infection.

In a further embodiment, the present invention provides methods andtherapeutic agents for treating diseases or disorders associated withabnormal or unwanted bone growth. For example, patients having thedisease known as Fibrodysplasia Ossificans Progressiva (FOP) grow anabnormal “second skeleton” that prevents any movement. Additionally,abnormal bone growth can occur after hip replacement surgery and thusruin the surgical outcome. This is a more common example of pathologicalbone growth and a situation in which the subject methods andcompositions may be therapeutically useful. The same methods andcompositions may also be useful for treating other forms of abnormalbone growth (e.g., pathological growth of bone following trauma, burnsor spinal cord injury), and for treating or preventing the undesirableconditions associated with the abnormal bone growth seen in connectionwith metastatic prostate cancer or osteosarcoma. Examples of thesetherapeutic agents include, but are not limited to, ActRII polypeptidesthat antagonize function of an ActRII ligand (e.g., BMP7), compoundsthat disrupt interaction between an ActRII and its ligand (e.g., BMP7),and antibodies that specifically bind to an ActRII receptor such that anActRII ligand (e.g., BMP7) cannot bind to the ActRII receptor.

In other embodiments, the present invention provides compositions andmethods for regulating body fat content in an animal and for treating orpreventing conditions related thereto, and particularly,health-compromising conditions related thereto. According to the presentinvention, to regulate (control) body weight can refer to reducing orincreasing body weight, reducing or increasing the rate of weight gain,or increasing or reducing the rate of weight loss, and also includesactively maintaining, or not significantly changing body weight (e.g.,against external or internal influences which may otherwise increase ordecrease body weight). One embodiment of the present invention relatesto regulating body weight by administering to an animal (e.g., a human)in need thereof an ActRII polypeptide.

In one specific embodiment, the present invention relates to methods andcompounds for reducing body weight and/or reducing weight gain in ananimal, and more particularly, for treating or ameliorating obesity inpatients at risk for or suffering from obesity. In another specificembodiment, the present invention is directed to methods and compoundsfor treating an animal that is unable to gain or retain weight (e.g., ananimal with a wasting syndrome). Such methods are effective to increasebody weight and/or mass, or to reduce weight and/or mass loss, or toimprove conditions associated with or caused by undesirably low (e.g.,unhealthy) body weight and/or mass.

In other embodiments, the subject ActRII polypeptides can be used toform pharmaceutical compositions that can be beneficially used toprevent, treat, or alleviate symptoms of a host of diseases involvingneurodegeneration. While not wishing to be bound by any particulartheory, the subject ActRII polypeptides may antagonize the inhibitoryfeedback mechanism mediated through the type I receptor ALK7, thusallowing new neuronal growth and differentiation. The subject ActRIIpolypeptides as pharmaceutical compositions can be beneficially used toprevent, treat, or alleviate symptoms of diseases withneurodegeneration, including Alzheimer's Disease (AD), Parkinson'sDisease (PD), Amyotrophic Lateral Sclerosis (ALS), and Huntington'sdisease (HD).

AD is a chronic, incurable, and unstoppable central nervous system (CNS)disorder that occurs gradually, resulting in memory loss, unusualbehavior, personality changes, and a decline in thinking abilities.These losses are related to the death of specific types of brain cellsand the breakdown of connections between them. AD has been described aschildhood development in reverse. In most people with AD, symptomsappear after the age 60. The earliest symptoms include loss of recentmemory, faulty judgment, and changes in personality. Later in thedisease, those with AD may forget how to do simple tasks like washingtheir hands. Eventually people with AD lose all reasoning abilities andbecome dependent on other people for their everyday care. Finally, thedisease becomes so debilitating that patients are bedridden andtypically develop coexisting illnesses. AD patients most commonly diefrom pneumonia, 8 to 20 years from disease onset.

PD is a chronic, incurable, and unstoppable CNS disorder that occursgradually and results in uncontrolled body movements, rigidity, tremor,and gait difficulties. These motor system problems are related to thedeath of brain cells in an area of the brain that produces dopamine, achemical that helps control muscle activity. In most people with PD,symptoms appear after age 50. The initial symptoms of PD are apronounced tremor affecting the extremities, notably in the hands orlips. Subsequent characteristic symptoms of PD are stiffness or slownessof movement, a shuffling walk, stooped posture, and impaired balance.There are wide ranging secondary symptoms such as memory loss, dementia,depression, emotional changes, swallowing difficulties, abnormal speech,sexual dysfunction, and bladder and bowel problems. These symptoms willbegin to interfere with routine activities, such as holding a fork orreading a newspaper. Finally, people with PD become so profoundlydisabled that they are bedridden. People with PD usually die frompneumonia.

ALS, also called Lou Gehrig's disease (motor neuron disease) is achronic, incurable, and unstoppable CNS disorder that attacks the motorneurons, components of the CNS that connect the brain to the skeletalmuscles. In ALS, the motor neurons deteriorate and eventually die, andthough a person's brain normally remains fully functioning and alert,the command to move never reaches the muscles. Most people who get ALSare between 40 and 70 years old. The first motor neurons that weaken arethose leading to the arms or legs. Those with ALS may have troublewalking, they may drop things, fall, slur their speech, and laugh or cryuncontrollably. Eventually the muscles in the limbs begin to atrophyfrom disuse. This muscle weakness will become debilitating and a personwill need a wheel chair or become unable to function out of bed. MostALS patients die from respiratory failure or from complications ofventilator assistance like pneumonia, 3-5 years from disease onset.

The causes of these neurological diseases have remained largely unknown.They are conventionally defined as distinct diseases, yet clearly showextraordinary similarities in basic processes and commonly demonstrateoverlapping symptoms far greater than would be expected by chance alone.Current disease definitions fail to properly deal with the issue ofoverlap and a new classification of the neurodegenerative disorders hasbeen called for.

HD is another neurodegenerative disease resulting from geneticallyprogrammed degeneration of neurons in certain areas of the brain. Thisdegeneration causes uncontrolled movements, loss of intellectualfaculties, and emotional disturbance. HD is a familial disease, passedfrom parent to child through a dominant mutation in the wild-type gene.Some early symptoms of HD are mood swings, depression, irritability ortrouble driving, learning new things, remembering a fact, or making adecision. As the disease progresses, concentration on intellectual tasksbecomes increasingly difficult and the patient may have difficultyfeeding himself or herself and swallowing. The rate of diseaseprogression and the age of onset vary from person to person.

Tay-Sachs disease and Sandhoff disease are glycolipid storage diseasescaused by the lack of lysosomal β-hexosaminidase (Gravel et al., in TheMetabolic Basis of Inherited Disease, eds. Scriver et al., McGraw-Hill,New York, pp. 2839-2879, 1995). In both disorders, GM2 ganglioside andrelated glycolipid substrates for β-hexosaminidase accumulate in thenervous system and trigger acute neurodegeneration. In the most severeforms, the onset of symptoms begins in early infancy. A precipitousneurodegenerative course then ensues, with affected infants exhibitingmotor dysfunction, seizure, visual loss, and deafness. Death usuallyoccurs by 2-5 years of age. Neuronal loss through an apoptotic mechanismhas been demonstrated (Huang et al., Hum. Mol. Genet. 6: 1879-1885,1997).

It is well known that apoptosis plays a role in AIDS pathogenesis in theimmune system. However, HIV-1 also induces neurological disease. Shi etal. (J. Clin. Invest. 98: 1979-1990, 1996) examined apoptosis induced byHIV-1 infection of the central nervous system (CNS) in an in vitro modeland in brain tissue from AIDS patients, and found that HIV-1 infectionof primary brain cultures induced apoptosis in neurons and astrocytes invitro. Apoptosis of neurons and astrocytes was also detected in braintissue from 10/11 AIDS patients, including 5/5 patients with HIV-1dementia and 4/5 nondemented patients.

Neuronal loss is also a salient feature of prion diseases, such asCreutzfeldt-Jakob disease in human, BSE in cattle (mad cow disease),Scrapie Disease in sheep and goats, and feline spongiform encephalopathy(FSE) in cats.

The subject ActRII polypeptides are also useful to prevent, treat, andalleviate symptoms of various PNS disorders, such as the ones describedbelow. The PNS is composed of the nerves that lead to or branch off fromthe CNS. The peripheral nerves handle a diverse array of functions inthe body, including sensory, motor, and autonomic functions. When anindividual has a peripheral neuropathy, nerves of the PNS have beendamaged. Nerve damage can arise from a number of causes, such asdisease, physical injury, poisoning, or malnutrition. These agents mayaffect either afferent or efferent nerves. Depending on the cause ofdamage, the nerve cell axon, its protective myelin sheath, or both maybe injured or destroyed.

The term “peripheral neuropathy” encompasses a wide range of disordersin which the nerves outside of the brain and spinal cord—peripheralnerves—have been damaged. Peripheral neuropathy may also be referred toas peripheral neuritis, or if many nerves are involved, the termspolyneuropathy or polyneuritis may be used.

Peripheral neuropathy is a widespread disorder, and there are manyunderlying causes. Some of these causes are common, such as diabetes,and others are extremely rare, such as acrylamide poisoning and certaininherited disorders. The most common worldwide cause of peripheralneuropathy is leprosy. Leprosy is caused by the bacterium Mycobacteriumleprae, which attacks the peripheral nerves of affected people.According to statistics gathered by the World Health Organization, anestimated 1.15 million people have leprosy worldwide.

Leprosy is extremely rare in the United States, where diabetes is themost commonly known cause of peripheral neuropathy. It has beenestimated that more than 17 million people in the United States andEurope have diabetes-related polyneuropathy. Many neuropathies areidiopathic—no known cause can be found. The most common of the inheritedperipheral neuropathies in the United States is Charcot-Marie-Toothdisease, which affects approximately 125,000 persons.

Another of the better known peripheral neuropathies is Guillain-Barresyndrome, which arises from complications associated with viralillnesses, such as cytomegalovirus, Epstein-Barr virus, and humanimmunodeficiency virus (HIV), or bacterial infection, includingCampylobacter jejuni and Lyme disease. The worldwide incidence rate isapproximately 1.7 cases per 100,000 people annually. Other well-knowncauses of peripheral neuropathies include chronic alcoholism, infectionof the varicella-zoster virus, botulism, and poliomyelitis. Peripheralneuropathy may develop as a primary symptom, or it may be due to anotherdisease. For example, peripheral neuropathy is only one symptom ofdiseases such as amyloid neuropathy, certain cancers, or inheritedneurologic disorders. Such diseases may affect the peripheral nervoussystem (PNS) and the central nervous system (CNS), as well as other bodytissues.

Other PNS diseases treatable with the subject ActRII polypeptidesinclude: Brachial Plexus Neuropathies (diseases of the cervical andfirst thoracic roots, nerve trunks, cords, and peripheral nervecomponents of the brachial plexus. Clinical manifestations includeregional pain, paresthesia; muscle weakness, and decreased sensation inthe upper extremity. These disorders may be associated with trauma,including birth injuries; thoracic outlet syndrome; neoplasms, neuritis,radiotherapy; and other conditions. See Adams et al., Principles ofNeurology, 6th ed, pp 1351-2); Diabetic Neuropathies (peripheral,autonomic, and cranial nerve disorders that are associated with diabetesmellitus). These conditions usually result from diabetic microvascularinjury involving small blood vessels that supply nerves (vasa nervorum).Relatively common conditions which may be associated with diabeticneuropathy include third nerve palsy; mononeuropathy; mononeuropathymultiplex; diabetic amyotrophy; a painful polyneuropathy; autonomicneuropathy; and thoracoabdominal neuropathy (see Adams et al.,Principles of Neurology, 6th ed, p 1325); mononeuropathies (disease ortrauma involving a single peripheral nerve in isolation, or out ofproportion to evidence of diffuse peripheral nerve dysfunction).Mononeuropathy multiplex refers to a condition characterized by multipleisolated nerve injuries. Mononeuropathies may result from a wide varietyof causes, including ischemia; traumatic injury; compression; connectivetissue diseases; cumulative trauma disorders; and other conditions);Neuralgia (intense or aching pain that occurs along the course ordistribution of a peripheral or cranial nerve); Peripheral NervousSystem Neoplasms (neoplasms which arise from peripheral nerve tissue.This includes neurofibromas; Schwannomas; granular cell tumors; andmalignant peripheral nerve sheath tumors. See DeVita Jr et al., Cancer:Principles and Practice of Oncology, 5th ed, pp 1750-1); NerveCompression Syndromes (mechanical compression of nerves or nerve rootsfrom internal or external causes. These may result in a conduction blockto nerve impulses, due to, for example, myelin sheath dysfunction, oraxonal loss. The nerve and nerve sheath injuries may be caused byischemia; inflammation; a direct mechanical effect; or Neuritis (ageneral term indicating inflammation of a peripheral or cranial nerve).Clinical manifestation may include pain; paresthesias; paresis; orhyperthesia; Polyneuropathies (diseases of multiple peripheral nerves).The various forms are categorized by the type of nerve affected (e.g.,sensory, motor, or autonomic), by the distribution of nerve injury(e.g., distal vs. proximal), by nerve component primarily affected(e.g., demyelinating vs. axonal), by etiology, or by pattern ofinheritance.

7. Pharmaceutical Compositions

In certain embodiments, compounds (e.g., ActRII polypeptides) of thepresent invention are formulated with a pharmaceutically acceptablecarrier. For example, an ActRII polypeptide can be administered alone oras a component of a pharmaceutical formulation (therapeuticcomposition). The subject compounds may be formulated for administrationin any convenient way for use in human or veterinary medicine.

In certain embodiments, the therapeutic method of the invention includesadministering the composition topically, systemically, or locally as animplant or device. When administered, the therapeutic composition foruse in this invention is, of course, in a pyrogen-free, physiologicallyacceptable form. Further, the composition may desirably be encapsulatedor injected in a viscous form for delivery to a target tissue site(e.g., bone, cartilage, muscle, fat or neuron), for example, a sitehaving a tissue damage. Topical administration may be suitable for woundhealing and tissue repair. Therapeutically useful agents other than theActRII polypeptides which may also optionally be included in thecomposition as described above, may alternatively or additionally, beadministered simultaneously or sequentially with the subject compounds(e.g., ActRII polypeptides) in the methods of the invention.

In certain embodiments, compositions of the present invention mayinclude a matrix capable of delivering one or more therapeutic compounds(e.g., ActRII polypeptides) to a target tissue site (e.g., bone,cartilage, muscle, fat or neuron), providing a structure for thedeveloping tissue and optimally capable of being resorbed into the body.For example, the matrix may provide slow release of the ActRIIpolypeptides. Such matrices may be formed of materials presently in usefor other implanted medical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the subjectcompositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid andpolyanhydrides. Other potential materials are biodegradable andbiologically well defined, such as bone or dermal collagen. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are non-biodegradable andchemically defined, such as sintered hydroxyapatite, bioglass,aluminates, or other ceramics. Matrices may be comprised of combinationsof any of the above mentioned types of material, such as polylactic acidand hydroxyapatite or collagen and tricalciumphosphate. The bioceramicsmay be altered in composition, such as in calcium-aluminate-phosphateand processing to alter pore size, particle size, particle shape, andbiodegradability.

In certain embodiments, methods of the invention can be administered fororally, e.g., in the form of capsules, cachets, pills, tablets, lozenges(using a flavored basis, usually sucrose and acacia or tragacanth),powders, granules, or as a solution or a suspension in an aqueous ornon-aqueous liquid, or as an oil-in-water or water-in-oil liquidemulsion, or as an elixir or syrup, or as pastilles (using an inertbase, such as gelatin and glycerin, or sucrose and acacia) and/or asmouth washes and the like, each containing a predetermined amount of anagent as an active ingredient. An agent may also be administered as abolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), one or more therapeuticcompounds of the present invention may be mixed with one or morepharmaceutically acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetylalcohol and glycerol monostearate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents such as ethoxylated isostearyl alcohols, polyoxyethylenesorbitol, and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Certain compositions disclosed herein may be administered topically,either to skin or to mucosal membranes. The topical formulations mayfurther include one or more of the wide variety of agents known to beeffective as skin or stratum corneum penetration enhancers. Examples ofthese are 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide,dimethylformamide, propylene glycol, methyl or isopropyl alcohol,dimethyl sulfoxide, and azone. Additional agents may further be includedto make the formulation cosmetically acceptable. Examples of these arefats, waxes, oils, dyes, fragrances, preservatives, stabilizers, andsurface active agents. Keratolytic agents such as those known in the artmay also be included. Examples are salicylic acid and sulfur.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches, and inhalants. The active compound may be mixed under sterileconditions with a pharmaceutically acceptable carrier, and with anypreservatives, buffers, or propellants which may be required. Theointments, pastes, creams and gels may contain, in addition to a subjectcompound of the invention (e.g., an ActRII polypeptide), excipients,such as animal and vegetable fats, oils, waxes, paraffins, starch,tragacanth, cellulose derivatives, polyethylene glycols, silicones,bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a subject compound,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

In certain embodiments, pharmaceutical compositions suitable forparenteral administration may comprise one or more ActRII polypeptidesin combination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents. Examples of suitable aqueous andnonaqueous carriers which may be employed in the pharmaceuticalcompositions of the invention include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol, and the like), andsuitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials, such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants.

The compositions of the invention may also contain adjuvants, such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption, such as aluminum monostearate andgelatin.

It is understood that the dosage regimen will be determined by theattending physician considering various factors which modify the actionof the subject compounds of the invention (e.g., ActRII polypeptides).The various factors include, but are not limited to, amount of boneweight desired to be formed, the site of bone damage, the condition ofthe damaged bone, the size of a wound, type of damaged tissue, thepatient's age, sex, and diet, the severity of any infection, time ofadministration, and other clinical factors. Optionally, the dosage mayvary with the type of matrix used in the reconstitution and the types ofcompounds in the composition. The addition of other known growth factorsto the final composition, may also effect the dosage. Progress can bemonitored by periodic assessment of bone growth and/or repair, forexample, X-rays, histomorphometric determinations, and tetracyclinelabeling.

In certain embodiments of the invention, one or more ActRII polypeptidescan be administered, together (simultaneously) or at different times(sequentially or overlapping). In addition, ActRII polypeptides can beadministered with another type of therapeutic agents, for example, acartilage-inducing agent, a bone-inducing agent, a muscle-inducingagent, a fat-reducing, or a neuron-inducing agent. The two types ofcompounds may be administered simultaneously or at different times. Itis expected that the ActRII polypeptides of the invention may act inconcert with or perhaps synergistically with another therapeutic agent.

In a specific example, a variety of osteogenic, cartilage-inducing andbone-inducing factors have been described, particularly bisphosphonates.See e.g., European Patent Application Nos. 148,155 and 169,016. Forexample, other factors that can be combined with the subject ActRIIpolypeptides include various growth factors such as epidermal growthfactor (EGF), platelet derived growth factor (PDGF), transforming growthfactors (TGF-α and TGF-β, and insulin-like growth factor (IGF).

In certain embodiments, the present invention also provides gene therapyfor the in vivo production of ActRII polypeptides. Such therapy wouldachieve its therapeutic effect by introduction of the ActRIIpolynucleotide sequences into cells or tissues having the disorders aslisted above. Delivery of ActRII polynucleotide sequences can beachieved using a recombinant expression vector such as a chimeric virusor a colloidal dispersion system. Preferred for therapeutic delivery ofActRII polynucleotide sequences is the use of targeted liposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. Retroviral vectors can be madetarget-specific by attaching, for example, a sugar, a glycolipid, or aprotein. Preferred targeting is accomplished by using an antibody. Thoseof skill in the art will recognize that specific polynucleotidesequences can be inserted into the retroviral genome or attached to aviral envelope to allow target specific delivery of the retroviralvector containing the ActRII polynucleotide. In one preferredembodiment, the vector is targeted to bone, cartilage, muscle or neuroncells/tissues.

Alternatively, tissue culture cells can be directly transfected withplasmids encoding the retroviral structural genes gag, pol and env, byconventional calcium phosphate transfection. These cells are thentransfected with the vector plasmid containing the genes of interest.The resulting cells release the retroviral vector into the culturemedium.

Another targeted delivery system for ActRII polynucleotides is acolloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. RNA, DNA andintact virions can be encapsulated within the aqueous interior and bedelivered to cells in a biologically active form (see e.g., Fraley, etal., Trends Biochem. Sci., 6:77, 1981). Methods for efficient genetransfer using a liposome vehicle, are known in the art, see e.g.,Mannino, et al., Biotechniques, 6:682, 1988. The composition of theliposome is usually a combination of phospholipids, usually incombination with steroids, especially cholesterol. Other phospholipidsor other lipids may also be used. The physical characteristics ofliposomes depend on pH, ionic strength, and the presence of divalentcations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Illustrative phospholipids include eggphosphatidylcholine, dipalmitoylphosphatidylcholine, anddistearoylphosphatidylcholine. The targeting of liposomes is alsopossible based on, for example, organ-specificity, cell-specificity, andorganelle-specificity and is known in the art.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments andembodiments of the present invention, and are not intended to limit theinvention.

Example 1 Generation of ActRIIB Mutants

Applicants generated a series of mutations in the extracellular domainof ActRIIB and produced these mutant proteins as soluble fusion proteinsbetween extracellular ActRIIB and an Fc domain. A co-crystal structureof Activin and extracellular ActRIIB did not show any role for the final(C-terminal) 15 amino acids (referred to as the “tail” herein) of theextracellular domain in ligand binding. This sequence failed to resolveon the crystal structure, suggesting that these residues are present ina flexible loop that did not pack uniformly in the crystal. Thompson etal. EMBO J. 2003 Apr. 1;22(7):1555-66. This sequence is also poorlyconserved between ActRIIB and ActRIIA. Accordingly, these residues wereomitted in the basic, or background, ActRIIB-Fc fusion construct.Additionally, position 64 in the background form is occupied by analanine, which is generally considered the “wild type” form, although aA64R allele occurs naturally. Thus, the background ActRIIB-Fc fusion hasthe sequence (Fc portion underlined)(SEQ ID NO:14):

SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHGYASWANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Surprisingly, as discussed below, the C-terminal tail was found toenhance activin and GDF-11 binding, thus a preferred version ofActRIIB-Fc has a sequence (Fc portion underlined)(SEQ ID NO:15):

SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK

Various mutations were introduced into the background ActRIIB-Fcprotein. Mutations were generated in ActRIIB extracellular domain by PCRmutagenesis. After PCR, fragments were purified thru Qiagen column,digested with SfoI and AgeI and gel purified. These fragments wereligated into expression vector pAID4 such that upon ligation it createdfusion chimera with human IgG1. Upon transformation into E. coli DH5alpha, colonies were picked and DNAs were isolated. All mutants weresequence verified.

All of the mutants were produced in HEK293T cells by transienttransfection. In summary, in a 500 ml spinner, HEK293T cells were set upat 6×10⁵ cells/ml in Freestyle (Invitrogen) media in 250 ml volume andgrown overnight. Next day, these cells were treated with DNA:PEI (1:1)complex at 0.5 ug/ml final DNA concentration. After 4 hrs, 250 ml mediawas added and cells were grown for 7 days. Conditioned media washarvested by spinning down the cells and concentrated.

All the mutants were purified over protein A column and eluted with lowpH (3.0) glycine buffer. After neutralization, these were dialyzedagainst PBS.

Mutants were also produced in CHO cells by similar methodology.

Mutants were tested in binding assays and bioassays described below.Proteins expressed in CHO cells and HEK293 cells were indistinguishablein the binding assays and bioassays.

Example 2 GDF-11 and Activin A Binding

Binding of ActRIIB-Fc proteins was tested in a BiaCore™ assay.

GDF-11 or Activin A (“ActA”) were immobilized on a BiaCore CM5 chipusing standard amine coupling procedure. The ActRIIB-Fc mutant orwild-type protein was loaded onto the system, and binding was measured.Results are summarized in Table 1, below.

TABLE 1 Soluble ActRIIB-Fc binding to GDF11 and Activin A (BiaCoreassay) 9779575_1 ActRIIB ActA GDF11 WT (64A) KD = 1.8e-7M KD = 2.6e-7M(+) (+) WT (64R) na KD = 8.6e-8M (+++) +15tail KD ~2.6 e-8M KD = 1.9e-8M(+++) (++++) E37A * * R40A − − D54A − * K55A ++ * R56A * * K74A KD =4.35e-9 M KD = 5.3e-9M +++++ +++++ K74Y * — K74F * — K74I * — W78A * *L79A + * D80K * * D80R * * D80A * * D80F * * D80G * * D80M * * D80N * *D80I * — F82A ++ − * No observed binding — <1/5 WT binding − ~1/2 WTbinding + WT ++ <2x increased binding +++ ~5x increased binding ++++~10x increased binding +++++ ~40x increased binding

As shown in Table 1, mutations had varying effects on ligand binding.The addition of the C-terminal 15 amino acids of the extracellulardomain caused a substantial increase in binding affinity for bothActivin A and GDF-11, and it is expected that this effect will translateto essentially all of the other mutations. Other mutations caused anoverall increase in ligand binding affinity, including the naturallyoccurring allele A64R and K74A. The R40A mutation caused a moderatedecrease in binding affinity for both Activin A and GDF-11. Manymutations abolished detectable binding to Activin A and GDF-11,including: E37A, R56A, W78A, D80K, D80R, D80A, D80G, D80F, D80M andD80N. Certain mutations caused a shift in selectivity. The followingmutations caused an increase in the ratio of GDF-11 to Activin Abinding: K74Y, K74F, K74I and D80I. The following mutations caused adecrease in the ratio of GDF-11 to Activin A binding: D54A, K55A, L79Aand F82A.

Example 3 Bioassay for GDF-11 and Activin-mediated Signaling

An A-204 Reporter Gene Assay was used to evaluate the effects ofActRIIB-Fc proteins on signaling by GDF-11 and Activin A. Cell line:Human Rhabdomyosarcoma (derived from muscle). Reporter vector:pGL3(CAGA)12 (Described in Dennler et al, 1998, EMBO 17: 3091-3100.) SeeFIG. 5. The CAGA12 motif is present in TGF-Beta responsive genes (PAI-1gene), so this vector is of general use for factors signaling throughSmad2 and 3.

Day 1: Split A-204 cells into 48-well plate.

Day 2: A-204 cells transfected with 10 ug pGL3(CAGA)12 orpGL3(CAGA)12(10 ug)+pRLCMV (1 ug) and Fugene.

Day 3: Add factors (diluted into medium+0.1% BSA). Inhibitors need to bepreincubated with Factors for 1 hr before adding to cells. 6 hrs later,cells rinsed with PBS, and lyse cells.

This is followed by a Luciferase assay. In the absence of anyinhibitors, Activin A showed 10 fold stimulation of reporter geneexpression and an ED50 ˜2 ng/ml. GDF-11: 16 fold stimulation, ED50: ˜1.5ng/ml.

As shown in FIG. 16, wild-type (background A64) ActRIIB-Fc inhibitsGDF-11 signaling in the A-204 Reporter Gene Assay. The background A64construct showed an inhibitory effect on GDF-11 activity. The A64Kmutation (also a naturally occurring form) caused a substantial increasein GDF-11 inhibition, and a combination of the A64K mutation with theaddition of the 15 C-terminal amino acids of the extracellular domain(the 15 amino acid “tail”) produced an even more potent inhibitor ofGDF-11 activity. As shown in FIG. 17, the background A64 constructshowed an inhibitory effect on Activin A activity. The K74A mutationcaused a substantial increase in Activin A inhibition. A control samplelacking Activin A showed no activity.

These data from the bioassay system correlate well with the bindingassays shown in Table 1 and demonstrate that the effects of the variousmutations translate to a biological system.

Example 4 ActRIIA-Fc Fusion Proteins

As shown in FIG. 14, ActRIIA and ActRIIB are highly conserved.Accordingly, most of the mutations tested in ActRIIB are expected tohave similar effects in ActRIIA. Thus, a background ActRIIA-Fc fusionmay be constructed with the following sequence (Fc portionunderlined)(SEQ ID NO:16):

AILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGOPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

As discussed below, the C-terminal tail was found to enhance activin andGDF-11 binding, thus a preferred version of ActRIIA-Fc has a sequence(Fc portion underlined)(SEQ ID NO:17):

AILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDLNCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK

Additional mutations, corresponding to those made in ActRIIB, may bemade in the background version of ActRIIA or the “tail” version ofActRIIA. The correspondence between ActRIIB and ActRIIA mutations isshown in Table 2 below.

Corresponding ActRIIA ActRIIB Mutant Functional Effect Mutant WT (64A)Background. WT is K65, so K65A mutation is expected to decrease bindingto all ligands. WT (64R) Increase binding to all ligands. K65,background. +15tail Increase binding to all ligands. +15 tail E37AEliminate detectable binding to all ligands. E38A R40A Decrease bindingto all ligands. R41A D54A Decrease GDF-11/Activin binding ratio. D55AK55A Decrease GDF-11/Activin binding ratio. K56A R56A Eliminatedetectable binding to all ligands. R57A K74A Increase binding to allligands. K75A K74Y Increase GDF-11/Activin binding ratio. K75Y K74FIncrease GDF-11/Activin binding ratio. K75F K74I Increase GDF-11/Activinbinding ratio. K75I W78A Eliminate detectable binding to all ligands.W79A L79A Decrease GDF-11/Activin binding ratio. L80A D80K Eliminatedetectable binding to all ligands. D81K D80R Eliminate detectablebinding to all ligands. D81R D80A Eliminate detectable binding to allligands. D81A D80F Eliminate detectable binding to all ligands. D81FD80G Eliminate detectable binding to all ligands. D81G D80M Eliminatedetectable binding to all ligands. D81M D80N Eliminate detectablebinding to all ligands. D81N D80I Increase GDF-11/Activin binding ratio.D81I F82A Decrease GDF-11/Activin binding ratio. I83A

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject matter have been discussed,the above specification is illustrative and not restrictive. Manyvariations will become apparent to those skilled in the art upon reviewof this specification and the claims below. The full scope of theinvention should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

1. An isolated polypeptide comprising an altered GDF11-binding domain ofan ActRII receptor having an increased selectivity for GDF11 versusactivin relative to a GDF11-binding domain of a wild-type receptor,wherein the amino acid sequence of the altered GDF11-binding domaincomprises: (i) the amino acid sequence of SEQ ID NO: 1 wherein the aminoacid residue at position 56 or 62 has been altered relative to thesequence of SEQ ID NO: 1, or (ii) the amino acid sequence of SEQ ID NO:2 wherein the amino acid residue at position 56 or 62 has been alteredrelative to the sequence of SEQ ID NO:
 2. 2. The polypeptide of claim 1,wherein the altered binding domain has a ratio of K_(d) for activinbinding to K_(d) for GDF11 binding that is at least 2 fold greater forthe altered binding domain relative to the ratio for the GDF11-bindingdomain of a wild-type receptor.
 3. The polypeptide of claim 1, whereinthe altered binding domain has a ratio of K_(d) for activin binding toK_(d) for GDF11 binding that is at least 5 fold greater for the alteredbinding domain relative to the ratio for the GDF11-binding domain of awild-type receptor.
 4. The polypeptide of claim 1, wherein the alteredbinding domain has a ratio of k_(d) for activin binding to k_(d) forGDF11 binding that is at least 10 fold greater for the altered bindingdomain relative to the ratio for the GDF11-binding domain of a wild-typereceptor.
 5. The polypeptide of claim 1, wherein the altered bindingdomain has a ratio of k_(d) for activin binding to k_(d) for GDF11binding that is at least 100 fold greater for the altered binding domainrelative to the ratio for the GDF11-binding domain of a wild-typereceptor.
 6. The polypeptide of claim 1, wherein the altered bindingdomain has a ratio of IC₅₀ for inhibiting activin to IC₅₀ for inhibitingGDF11 that is at least 2 fold greater for the altered binding domainthan the GDF11-binding domain of a wild-type receptor.
 7. Thepolypeptide of claim 1, wherein the altered binding domain has a ratioof IC₅₀ for inhibiting activin to IC₅₀ for inhibiting GDF11 that is atleast 5 fold greater for the altered binding domain than theGDF11-binding domain of a wild-type receptor.
 8. The polypeptide ofclaim 1, wherein the altered binding domain has a ratio of IC₅₀ forinhibiting activin to IC₅₀ for inhibiting GDF11 that is at least 10 foldgreater for the altered binding domain than the GDF11-binding domain ofa wild-type receptor.
 9. The polypeptide of claim 1, wherein the alteredbinding domain has a ratio of IC₅₀ for inhibiting activin to IC₅₀ forinhibiting GDF11 that is at least 100 fold greater for the alteredbinding domain than the GDF11-binding domain of a wild-type receptor.10. The polypeptide of claim 1, which inhibits GDF11 with an IC₅₀ atleast 2 times less than the IC₅₀ of the polypeptide for inhibitingactivin.
 11. The polypeptide of claim 1, which inhibits GDF11 with anIC₅₀ at least 5 times less than the IC₅₀ of the polypeptide forinhibiting activin.
 12. The polypeptide of claim 1, which inhibits GDF11with an IC₅₀ at least 10 times less than the IC₅₀ of the polypeptide forinhibiting activin.
 13. The polypeptide of claim 1, which inhibits GDF11with an IC₅₀ at least 100 times less than the IC₅₀ of the polypeptidefor inhibiting activin.
 14. The polypeptide of claim 1, wherein theGDF11 polypeptide is a fusion protein.
 15. The polypeptide of claim 14,wherein said altered GDF11-binding domain of an ActRII receptor is fusedto an IgG Fc domain.
 16. The polypeptide of claim 15, wherein said IgGFc domain comprises one or more mutations.
 17. The polypeptide of claim16, wherein the Fc domain has reduced ability to bind to the Fcγreceptor relative to a wild-type Fc domain.
 18. The polypeptide of claim16, wherein the Fc domain has increase ability to bind to the MHC classI-related Fc-receptor (FcRN) relative to a wild-type Fc domain.
 19. Thepolypeptide of claim 15, wherein the Fc domain has a mutation atresidues selected from the group consisting of: Asp-265, lysine 322, andAsn-434.
 20. The polypeptide of claim 16, wherein the mutation is shownin FIG.
 12. 21. The polypeptide of claim 15, wherein the IgG Fc domainhas an amino acid sequence as set forth in SEQ ID NO:
 13. 22. Thepolypeptide of claim 1, wherein the altered GDF11-binding domaincomprises the amino acid sequence of SEQ ID NO: 1 wherein the amino acidat position 56 has been changed to a tyrosine.
 23. The polypeptide ofclaim 1, wherein the altered GDF11-binding domain comprises the aminoacid sequence of SEQ ID NO: 1 wherein the amino acid at position 56 hasbeen changed to a phenylalanine.
 24. The polypeptide of claim 1, whereinthe altered GDF11-binding domain comprises the amino acid sequence ofSEQ ID NO: 1 wherein the amino acid at position 56 has been changed to aisoleucine.
 25. The polypeptide of claim 1, wherein the alteredGDF11-binding domain comprises the amino acid sequence of SEQ ID NO: 1wherein the amino acid at position 62 has been changed to an isoleucine.26. The polypeptide of claim 1, wherein the altered GDF11-binding domaincomprises the amino acid sequence of SEQ ID NO: 2 wherein the amino acidat position 56 has been changed to a tyrosine.
 27. The polypeptide ofclaim 1, wherein the altered GDF11-binding domain comprises the aminoacid sequence of SEQ ID NO: 2 wherein the amino acid at position 56 hasbeen changed to a phenylalanine.
 28. The polypeptide of claim 1, whereinthe altered GDF11-binding domain comprises the amino acid sequence ofSEQ ID NO: 2 wherein the amino acid at position 56 has been changed to aisoleucine.
 29. The polypeptide of claim 1, wherein the alteredGDF11-binding domain comprises the amino acid sequence of SEQ ID NO: 2wherein the amino acid at position 62 has been changed to an isoleucine.